U.S. patent application number 16/124304 was filed with the patent office on 2019-01-03 for digital measurements from targeted sequencing.
The applicant listed for this patent is NuGEN Technologies, Inc.. Invention is credited to Douglas A. Amorese, Stephanie C. Huelga, Benjamin G. Schroeder, Jonathan Scolnick.
Application Number | 20190005193 16/124304 |
Document ID | / |
Family ID | 55264574 |
Filed Date | 2019-01-03 |
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United States Patent
Application |
20190005193 |
Kind Code |
A1 |
Scolnick; Jonathan ; et
al. |
January 3, 2019 |
DIGITAL MEASUREMENTS FROM TARGETED SEQUENCING
Abstract
Disclosed herein are methods, compositions and kits for
quantitating one or more specific nucleic acids within a plurality
of nucleic acids. In some embodiments, a sequencing library is
constructed from enriched probe extension products specific for the
specific nucleic acids and sequenced. In some embodiments, the
resulting reads are used for removing duplicate reads. In some
embodiments, counting of verified probes is used to quantitate or
determine the number of specific nucleic acid molecules in the
starting nucleic acid sample.
Inventors: |
Scolnick; Jonathan; (San
Francisco, CA) ; Schroeder; Benjamin G.; (San Mateo,
CA) ; Amorese; Douglas A.; (Los Altos, CA) ;
Huelga; Stephanie C.; (Belmont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NuGEN Technologies, Inc. |
San Carlos |
CA |
US |
|
|
Family ID: |
55264574 |
Appl. No.: |
16/124304 |
Filed: |
September 7, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14820250 |
Aug 6, 2015 |
10102337 |
|
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16124304 |
|
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62034043 |
Aug 6, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G16B 50/00 20190201;
C12Q 1/689 20130101; C12Q 1/6886 20130101; G16B 30/00 20190201;
C12Q 1/6806 20130101; C12Q 2600/158 20130101; C12Q 1/689 20130101;
C12Q 2525/191 20130101; C12Q 2535/122 20130101; C12Q 2537/165
20130101; C12Q 1/6806 20130101; C12Q 2525/191 20130101; C12Q
2535/122 20130101; C12Q 2537/165 20130101 |
International
Class: |
G06F 19/22 20060101
G06F019/22; C12Q 1/6886 20060101 C12Q001/6886; G06F 19/28 20060101
G06F019/28; C12Q 1/689 20060101 C12Q001/689; C12Q 1/6806 20060101
C12Q001/6806 |
Claims
1. A method for quantitating a plurality of specific nucleic acid
molecules in a composition comprising: a. generating a plurality of
probe extension products, wherein each probe extension product
comprises a probe sequence that is complementary to a probe target
region within a specific nucleic acid molecule; b. sequencing the
plurality of probe extension products to generate a sequence for
each of the plurality of probe extension products; c. aligning the
sequence of each of the plurality of probe extension products to a
reference sequence database, wherein the reference sequence
database comprises probe sequences; and d. determining the number
of alignments for the sequence of each probe extension product with
a sequence in the reference sequence database, wherein the number
of alignments indicates the quantity of each of the specific
nucleic acid molecule that the probe of the probe extension product
is complementary to.
2. A method for quantitating a plurality of specific nucleic acid
molecules comprising: a. generating a plurality of probe extension
products, wherein each probe extension product comprises (i) a
first adapter, and (ii) a probe sequence complementary to a probe
target region within a specific nucleic acid molecule; b.
sequencing the plurality of probe extension products to generate
sequence data comprising a sequence for each of the plurality of
probe extension products; c. identifying the presence of the probe
sequence of each probe extension product within the sequence data;
and d. determining the number of each of the probe sequences within
the plurality of probe extension products, wherein the number of
each of the probe sequences indicates the quantity of each of the
plurality of specific nucleic acid molecules to which each of the
probes sequences is complementary to.
3. A method for quantitating a plurality of specific nucleic acid
molecules within a plurality of nucleic acid molecules comprising:
a. appending a first adaptor sequence to a 5' end to each of a
plurality of nucleic acid molecules; b. hybridizing a plurality of
probes to the plurality of specific nucleic acid molecules, wherein
each probe is complementary to a probe target region within a
specific nucleic acid molecule; c. extending each probe into the
appended first adaptor sequence to generate a plurality of probe
extension products having the first adaptor sequence and a second
adaptor sequence to produce a plurality of probe extension
products; d. sequencing the plurality of probe extension products
to generate sequence data for each of the plurality of probe
extension products; e. aligning the sequence for each of the
plurality of probe extension products to a pre-determined sequence
within a reference copy of a probe database, wherein said
pre-determined sequence is specific to each probe; and f.
determining the number of each probe sequence aligned to its
pre-determined sequence, wherein the number indicates the quantity
of the specific nucleic acids molecule to which the probe is
complementary to.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/820,250, filed Aug. 6, 2015, which application claims the
benefit of U.S. Provisional Application Ser. No. 62/034,043, filed
Aug. 6, 2014, both incorporated by reference.
FIELD OF THE INVENTION
[0002] The present teachings relate to the use of targeted nucleic
acid sequencing that result in digital measurements for gene
expression and copy number variation.
BACKGROUND OF THE INVENTION
[0003] Molecular methods that provide digital counts of a specific
nucleic acid(s) are of interest to the research and clinical
community. These methods can be used to discretely measure gene
expression (digital gene expression or DGE) or copy number
variation (CNV). The precision measurements that can be obtained by
digital readouts provides higher confidence in data compared to
microarray technology and allows researchers to identify smaller
differences between samples or similarly, differences within
subsets of cells such as in a tumor biopsy as well as determining
cell to cell variations.
[0004] However there is still a need for different methods for
selective target quantitation that allow for high throughput
analysis of transcriptome and genomic regions of interest without
specialized instrumentation. The methods, compositions and kits
disclosed herein fulfill these needs and provide related
advantages.
SUMMARY OF THE INVENTION
[0005] In one aspect, disclosed is a method for quantitating a
plurality of specific nucleic acids within a plurality of nucleic
acids comprising: a. generating a sequencing library of a plurality
of probe extension products, wherein each probe extension product
can be derived from extending a probe complementary to and
hybridized to a probe target region within a specific nucleic acid
sequence; b. sequencing the library comprising the plurality of
probe extension products to generate sequence data for the
plurality of probe extension products; and c. counting each of the
aligned sequences, wherein the number of alignments indicates the
quantity of each of the corresponding specific nucleic acid
molecules, within the plurality of nucleic acids.
[0006] In one aspect, disclosed in a method for quantitating a
plurality of specific nucleic acid molecules in a composition
comprising: a. generating a plurality of probe extension products,
wherein each probe extension product comprises a probe sequence
that is complementary to a probe target region within a specific
nucleic acid molecule; b. sequencing the plurality of probe
extension products to generate a sequence for each of the plurality
of probe extension products; c. aligning the sequence of each of
the plurality of probe extension products to a reference sequence
database, wherein the reference sequence database comprises probe
sequences; and d. determining the number of alignments for the
sequence of each probe extension product with a sequence in the
reference sequence database, wherein the number of alignments
indicates the quantity of each of the specific nucleic acid
molecule that the probe of the probe extension product is
complementary to.
[0007] In one aspect, disclosed is a method for quantitating a
plurality of specific nucleic acids within a plurality of nucleic
acids comprising: a. generating a sequencing library of a plurality
of probe extension products, wherein each probe extension product
comprises a first adapter attached to the 5' end of each probe
extension product, wherein each probe extension product can be
derived from extending a probe complementary to and hybridized to a
probe target region within a specific nucleic acid sequence; b.
sequencing the library to generate sequence data for the plurality
of probe extension products; and c. identifying the presence of the
probe sequence within the sequence data and counting each probe
sequence within the plurality of probe extension products, wherein
the number of probes counted indicates the quantity of each of the
plurality of specific nucleic acid molecules within the plurality
of nucleic acids.
[0008] In one aspect, disclosed is a method for quantitating a
plurality of specific nucleic acid molecules comprising: a.
generating a plurality of probe extension products, wherein each
probe extension product comprises (i) a first adapter, and (ii) a
probe sequence complementary to to a probe target region within a
specific nucleic acid molecule; b. sequencing the plurality of
probe extension products to generate sequence data comprising a
sequence for each of the plurality of probe extension products; c.
identifying the presence of the probe sequence of each probe
extension product within the sequence data; and d. determining the
number of each of the probe sequences within the plurality of probe
extension products, wherein the number of each of the probe
sequences indicates the quantity of each of the plurality of
specific nucleic acid molecules to which each of the probes
sequences is complementary to.
[0009] In one aspect, disclosed is a method for quantitating a
plurality of specific nucleic acids within a plurality of nucleic
acids comprising: a. appending a first adaptor sequence to a 5' end
of a plurality of nucleic acids; b. hybridizing a plurality of
probes, wherein each probe is complementary to a probe target
region within a specific nucleic acid within the plurality of
specific nucleic acids; c. extending each probe into the appended
first adaptor sequence to generate a plurality of probe extension
products having the first adaptor sequence and a second adaptor
sequence; d. generating a sequencing library comprising the
plurality of probe extension products; e. sequencing the library,
wherein sequence data is obtained for each of the plurality of
probe extension products; f. aligning the sequence data for each of
the plurality of probe extension products to a pre-determined
sequence within a reference copy of a probe database, wherein said
pre-determined sequence is specific to each probe; and g. counting
each probe sequence aligned to its pre-determined sequence, wherein
the number of counts for each probe specific for its specific
nucleic acid indicates the quantity of each of the specific nucleic
acids molecules within the plurality of specific nucleic acids
within the plurality of nucleic acids.
[0010] In one aspect, disclosed is a method for quantifying a
plurality of specific nucleic acid molecules within a plurality of
nucleic acid molecules comprising: a. appending a first adaptor
sequence to a 5' end to each of a plurality of nucleic acid
molecules; b. hybridizing a plurality of probes to the plurality of
specific nucleic acid molecules, wherein each probe is
complementary to a probe target region within a specific nucleic
acid molecule; c. extending each probe into the appended first
adaptor sequence to generate a plurality of probe extension
products having the first adaptor sequence and a second adaptor
sequence to produce a plurality of probe extension products; d.
sequencing the plurality of probe extension products to generate
sequence data for each of the plurality of probe extension
products; e. aligning the sequence for each of the plurality of
probe extension products to a pre-determined sequence within a
reference copy of a probe database, wherein said pre-determined
sequence is specific to each probe; and f. determining the number
of each probe sequence aligned to its pre-determined sequence,
wherein the number indicates the quantity of the specific nucleic
acids molecule to which the probe is complementary to.
[0011] In one aspect, disclosed is a method for quantitating a
plurality of specific nucleic acids within a plurality of nucleic
acids comprising: a. extending a plurality of hybridized probes,
wherein each probe is complementary to a probe target region within
a specific nucleic acid within the plurality of specific nucleic
acids and each probe has a 5' first adaptor; b. appending a second
adaptor sequence to the double-stranded end of the plurality of
probe extension products to generate a sequencing library; c.
sequencing the library, wherein sequence data can be obtained for
each of the plurality of probe extension products; and d. counting
each probe sequence corresponding to each probe target region,
wherein the number of counts for each probe specific for its
specific nucleic acid indicates the quantity of each of the
specific nucleic acids molecules within the plurality of specific
nucleic acids within the plurality of nucleic acids.
[0012] In one aspect, disclosed is a method for quantitating a
plurality of specific nucleic acid molecules comprising: a.
extending a plurality of probes, wherein each probe is hybridized
to a probe target region within a specific nucleic acid molecule
within the plurality of specific nucleic acid molecules and each
probe has a first adaptor at its 5' end to generate a plurality of
extension products; b. appending a second adaptor to the
double-stranded end of the plurality of probe extension products;
c. sequencing the plurality of probe extension products to generate
sequence data for each of the probe extension products; and d.
determining the number of each probe that hybridized to a probe
target region, wherein the number indicates the quantity of each of
the specific nucleic acid molecules comprising the probe target
region.
[0013] In one aspect, disclosed is a method for quantitating a
plurality of specific nucleic acids within a plurality of nucleic
acids comprising: a. hybridizing a plurality of probes, wherein
each probe is complementary to a probe target region within a
specific nucleic acid within the plurality of specific nucleic
acids and each probe has a 5' first adaptor; b. extending each
probe to generate a plurality of probe extension products having
the first adaptor sequence; c. appending a second adaptor sequence
to the double-stranded end of the plurality of probe extension
products; d. generating a sequencing library comprising the
plurality of probe extension products; e. sequencing the library,
wherein sequence data can be obtained for each of the plurality of
probe extension products; f. aligning the sequence data for each of
the plurality of probe extension products to a pre-determined
sequence within a probe database, wherein said pre-determined
sequence is specific to each probe; and g. counting each probe
sequence aligned to the probe target region, wherein the number of
counts for each probe specific for its specific nucleic acid
indicates the quantity of each of the specific nucleic acids
molecules within the plurality of specific nucleic acids within the
plurality of nucleic acids.
[0014] In one aspect, disclosed is a method for quantitating a
plurality of specific nucleic acid molecules in a composition
comprising: a. hybridizing a plurality of probes to a probe target
region within a specific nucleic acid molecule, wherein each probe
has a first adaptor at its 5' end; b. extending each probe to
generate a plurality of probe extension products comprising the
first adaptor sequence; c. appending a second adaptor sequence to
the double-stranded end of the plurality of probe extension
products; d. sequencing the plurality of probe extension products
to generate sequence for each of the plurality of probe extension
products; e. aligning the sequence for each of the plurality of
probe extension products to a pre-determined sequence within a
probe database, wherein said probe database comprises a plurality
of pre-determined sequences, wherein each pre-determined sequence
is specific to a probe; and f. determining the number of alignments
for the sequence of each probe extension product to a
pre-determined sequence within the sequencing database, wherein the
number of of alignments indicates the quantity of each of the
specific nucleic acids molecules to which the probe hybridizes
to.
[0015] In some embodiments, the sequence data or sequenced
plurality of probe extension products comprise at least one of a
forward read, an index read and a reverse read. In some
embodiments, the reverse read comprises the probe target region. In
some embodiments, specificity that each probe has annealed to its
respective probe target region sequence within its respective
specific nucleic acid can be verified. In some embodiments, the
sequence data or sequenced plurality of probe extension products
can be mapped to coordinates of a genome or a transcriptome
database and/or the sequence data or sequenced plurality of probe
extension products can be aligned to a reference copy of a probe
database to verify intended probe annealing and extension. In some
embodiments, the sequence data or sequenced plurality of probe
extension products can be mapped to coordinates of a genome or a
transcriptome database. In some embodiments, the reverse read or
the forward read comprises the probe target region. In some
embodiments, the sequence data or sequenced plurality of probe
extension products for the forward and reverse reads can be mapped
for the plurality of specific nucleic acids and the sequence data
or sequenced plurality of probe extension products for the index
read can identify at least one of the barcode sequence and the
n-random sequence. In some embodiments, the combination of the
forward read map coordinates and the index read n-random bases
determine PCR duplicates for each probe extension product and
sequences having the same forward read coordinates and the same
n-random base sequence can be identified as duplicates,
consolidated and counted as a single specific nucleic acid
molecule; and wherein sequences with the same forward read
coordinates but different n-random base sequences can be each
counted as a distinct specific nucleic acid molecule.
[0016] In some embodiments, the forward reads and corresponding
reverse reads can be pair end aligned. In some embodiments,
following duplicate consolidation, the number of reverse reads or
forward reads counted for each probe sequence generates a value
that represents the number of molecules for each starting specific
nucleic acid molecule within the plurality of specific nucleic
acids. In some embodiments, the genome is selected from the group
consisting of a mammalian, bacterial, viral, rickettsial or plant
genome or transcriptome. In some embodiments, the plurality of
specific nucleic acids have undergone end repair prior to appending
the first adaptor. In some embodiments, the end repair is blunt end
repair. In some embodiments, the probe can be extended by a
polymerase selected from the group consisting of a DNA polymerase,
an RNA polymerase or a reverse transcriptase.
[0017] In some embodiments, prior to generating the sequencing
library the plurality of probe extension products can be amplified
or optionally are amplified. In some embodiments, the probe
extension product can be treated with a restriction endonuclease or
undergoes blunt end/end repair prior to addition of the second
adaptor. In some embodiments, wherein extension of the probe
extension product further comprises addition of a first adaptor. In
some embodiments, amplification of the probe extension product
further comprises attachment of a flow cell sequence to each end of
the amplification product. In some embodiments, the restriction
endonuclease treated probe extension product yields a forward read
with a common end. In some embodiments, the sequence data or
sequenced plurality of probe extension products can be mapped to
coordinates of a genome or transcriptome to verify intended probe
annealing and extension. In some embodiments, the sequence data or
sequenced plurality of probe extension products can be aligned to a
reference copy of a probe database to verify intended probe
annealing. In some embodiments, reverse read sequences or the
forward read sequences can be binned and counted according to which
probe sequence they represent, wherein the number of times each
probe is represented can be a measure of the number of times the
starting specific nucleic acid molecule is present in the original
sample. In some embodiments, the forward read comprises at least a
portion of the specific nucleic acid sequence that can include at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, at least 11, at least 12, at least 13, at least 14,
at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, or at least 25 bases of the specific nucleic acid
sequence.
[0018] In some embodiments, the first adaptor sequence or the
second adaptor sequence comprises at least one of an index sequence
priming site, an index nucleotide sequence, an n-random nucleotide
sequence, a forward read priming site, and a reverse read priming
site, and combinations thereof. In some embodiments, the second
adaptor sequence or the first adaptor sequence comprises at least
one of a forward read priming site, a reverse read priming site and
a linker sequence, and combinations thereof. In some embodiments,
the 5' first adaptor can be common to each probe extension product.
In some embodiments, the 5' tail sequence can include a second
adaptor sequence. In some embodiments, amplification of the probe
extension product yields attachment of a flow cell sequence to each
end of the amplification product.
[0019] In some embodiments, the index read comprises at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, or at least 15 bases of
index nucleotide sequence and the n-random base sequence. In some
embodiments, the index read comprises at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, or at least 10 bases
of the n-random bases and the index nucleotide sequence. In some
embodiments, the index read comprises at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, or at least 10 bases
of the n-random bases and optionally, the index nucleotide
sequence. In some embodiments, the n-random base nucleotide
sequence comprises at least 1, at least 2, at least 3, at least 4,
at least 5, at least 6, at least 7, at least 8, at least 9, or at
least 10 nucleotides. In some embodiments, the index nucleotide
sequence further comprises a barcode sequence.
[0020] In some embodiments, the reverse read comprises at least one
of a probe sequence and a portion of a specific nucleic acid
sequence and the combination thereof. In some embodiments, the
reverse read comprises at least 5, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45, at least 50, at
least 55, or at least 60 bases of probe sequence. In some
embodiments, the reverse read comprises at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, or at least
20 bases of specific nucleic acid sequence 3' to the probe
sequence.
[0021] In a further aspect, disclosed is a composition of probe
extension products produced and/or amplified by the disclosed
methods.
[0022] In yet a further aspect, the plurality of nucleic acids can
be derived from a sample selected from the group consisting of a
tissue, an organ, a single cell, a tumor, a specimen of an organic
fluid taken from a patient, freely circulating nucleic acids, a
fungus, a prokaryotic organism, and a virus. In some embodiments,
the patient can be known or suspected of having a tumor. In some
embodiments, the organic fluid contains at least one circulating
tumor cell (CTC) or a disseminated tumor cell (CTD). In some
embodiments, the patient can be known or suspected of having a
viral infection that can be a communicable infection or a
communicable disease.
[0023] In some embodiments compositions of the present disclosure
comprise a plurality of nucleic acid molecules. In some
embodiments, each probe extension product is an extension product
of a probe complementary to a probe target region within a specific
nucleic acid molecule.
[0024] In yet a further aspect, disclosed is a kit for digital
measurement of nucleic acid molecules comprising at least one or
more of: an oligonucleotide adaptor; a probe complementary to a
portion of a probe target region sequence; a primer complementary
to said adaptor sequence; a primer complementary to a portion of
the probe sequence; a ligase; a polymerase; and instructions for
use of the kit. In yet a further aspect, disclosed is a kit for
digital measurement of nucleic acid molecules comprising one or
more aspects of the present disclosure.
[0025] In some embodiments, methods, compositions, and kits of the
present disclosure comprise one or more aspects disclosed in Li et
al. 2012. Bioinformatics. 28(10):1307-1313; Bellos et al. 2014.
Nucleic Acids Res. 42(20):e158; Jiang et al. 2015. Nucleic Acids
Res. 43(6):e39; Xi et al. 2011. Proc. Natl. Acad. Sci.
108(46):1128-1136; Fromer and Purcell. 2014. Curr. Protoc. Hum.
Genet. 81:7.21.1-7.23.21; Sathirapongsasuti et al. 2011.
Bioinformatics. 31(15):1-8; Krumm et al. 2012. Genome Res.
22(8):1525-1532; Plagnol et al. 2012. Bioinformatics.
28(21):2747-2754.
INCORPORATION BY REFERENCE
[0026] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
[0027] Pending applications U.S. Ser. No. 13/750,768, U.S. Ser. No.
14/030,761, and U.S. Ser. No. 61/903,826 are incorporated by
reference in their entirety herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] A better understanding of the novel features and advantages
of the disclosed invention can be obtained by reference to the
following description that sets forth illustrative embodiments, in
which the principles of the disclosed invention are utilized, and
the accompanying drawings of which:
[0029] FIG. 1 is a flow chart illustrating embodiments of library
generation disclosed herein using gDNA.
[0030] FIG. 2 is a flow chart illustrating embodiments of library
generation disclosed herein using cDNA.
[0031] FIG. 3 is a flow chart illustrating embodiments of library
generation disclosed herein using double-stranded gDNA.
[0032] FIG. 4 is a flow chart illustrating embodiments of library
generation disclosed herein using double-stranded gDNA.
[0033] FIG. 5 illustrates embodiments disclosed herein for
constructing a sequencing library and regions of sequencing
reads.
[0034] FIGS. 6A-6C illustrate embodiments disclosed herein for
removing duplicate reads from sequencing data. FIG. 6A--forward
read, FIG. 6B--index read, FIG. 6C--reverse read.
[0035] FIGS. 7A-7C illustrate embodiments disclosed herein for
identifying the regions sequenced to obtain sequence data: FIG.
7A--forward read, FIG. 7B--index read, FIG. 7C--reverse read.
[0036] FIGS. 8A-8C illustrate embodiments disclosed herein for
identifying the regions sequenced to obtain sequence data: FIG.
8A--Probe containing sequence read, FIG. 8B--Specific nucleic acid
sequencing read, FIG. 8C--indexing sequencing read comprising at
least one of an index base read and an n-random base read or a
combination thereof.
[0037] FIG. 9 graphically illustrates embodiments disclosed herein
for generation of sequencing libraries and subsequent digital
quantification.
[0038] FIG. 10 graphically illustrates embodiments disclosed herein
for using a sequencing library for NGS sequencing and analyzing
sequence data for digital quantification.
[0039] FIG. 11A, FIG. 11B, and FIG. 11C graphically illustrate the
plot of gene abundance at the RNA level in a panel of 95 genes in
chromosomal order. Genes colored with a dot and appearing below the
zero-value line are significantly downregulated, and genes colored
with a dot and appearing above the zero-value line are
significantly upregulated. Error bars reflect the standard
deviation in both the DNA and RNA data. FIG. 11A shows genes in
chromosome order from 1 to 6. FIG. 11B shows genes in chromosome
order from 7 to 15. FIG. 11C shows genes in chromosome order from
16 to X.
[0040] FIG. 12A, FIG. 12B, and FIG. 12C graphically illustrate the
plot of measured levels for all genes in the 509 gene panel sorted
in chromosomal order. Genes with copy number changes are colored
with a dot and appear above the one-value line. Error bars are
reflective of combined variation in the probe counts of the sample
and control datasets. FIG. 12A shows genes in chromosome order from
1 to 6. FIG. 12B shows genes in chromosome order from 7 to 14. FIG.
12C shows genes in chromosome order from 15 to X.
DETAILED DESCRIPTION
[0041] This disclosure describes a method for targeted nucleic acid
sequencing resulting in digital measurements. Examples of where
these digital measurements are useful are in digital gene
expression and copy number variation. Starting material can be
nucleic acid, DNA, RNA, cDNA, or double stranded cDNA. The
disclosed methods, compositions and kits describe utilizing a
complementary probe hybridized to its probe target region to
generate probe extension products derived from the probe target
region. The probe extension products are used for target enrichment
and library generation proceeding high throughput sequencing.
Analysis of the sequencing data provides digital measurements of
transcriptome gene expression or genomic DNA copy number
variation.
[0042] Targeting probes are hybridized to a specific nucleic acid
and extended with a polymerase using the target enrichment kit sold
under the trademark OVATION by NuGEN. Paired end sequencing can be
performed on the resulting enriched library. Reads are mapped to
the genome or transcriptome and PCR duplicate reads are identified
(described in patent application U.S. Ser. No. 61/903,826). Probe
sequences are then counted for how many times they appear in the
de-duplicated sequencing dataset as a measure of the number of
copies of the original nucleic acid that were present in the
starting sample. Using probe sequence counts instead of random
sequence simplifies copy number analysis because precisely the same
sequences are being assessed across different samples for each
digital measurement. This can serve to normalize for such factors
as gene length, which can change between samples due to alternative
exon usage, as well as reducing known problems with sequencing read
mapping to the genome or transcriptome.
[0043] The methods of the disclosed invention can be used with
various applications for genetic sample analysis including but not
limited to RNA-Seq analysis, digital gene expression, genotyping,
copy number variation determination and whole genome
amplification.
[0044] Unless otherwise specified, terms and symbols of
biochemistry, nucleic acid chemistry, molecular biology and
molecular genetics follow those of standard treaties and texts in
the field, for example, Sambrook et al, Molecular Cloning: A
Laboratory Manual, 2.sup.nd Edition (Cold Spring Harbor Laboratory,
1989); Kornberg and Baker, DNA Replication, Second Edition (W.H.
Freeman, New York, 1992); Gaits, ed., Oligonucleotide Synthesis: A
Practical Approach (IRL Press, Oxford, 1984); Lehninger,
Biochemistry, Second Edition (Worth Publishers, New York, 1975);
Eckstein, ed., Oligonucleotides and Analogs: A Practical Approach
(Oxford University Press, New York, 1991); and the like.
[0045] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a polymerase" can refer to one agent or to mixtures of such
agents, and reference to "the method" includes reference to
equivalent steps and/or methods known to those skilled in the art,
and so forth.
[0046] Additionally, to facilitate understanding, disclosed are a
number of terms as defined herein.
[0047] The term "adaptor", as used herein, can refer to an
oligonucleotide of known sequence, the attachment of which to a
specific nucleic acid sequence or a target polynucleotide strand of
interest enables the generation of amplification-ready products of
the specific nucleic acid or the target polynucleotide strand of
interest. The specific nucleic acid samples can be fragmented or
not prior to the addition of at least one adaptor.
[0048] Various adaptor designs are envisioned which are suitable
for generation of amplification-ready products of specific sequence
regions/strands of interest. For example, when double stranded
adaptors are used, the two strands of the adaptor can be
self-complementary, non-complementary or partially complementary.
Adaptors can contain at least a partial forward sequence priming
site and a random sequence.
[0049] In some embodiments, adaptors comprise an additional
identifier sequence, e.g., a barcode sequence. As used herein, the
term "barcode" can refer to a known nucleic acid sequence that
allows some feature of a polynucleotide with which the barcode is
associated to be identified. In some embodiments, the feature of
the polynucleotide to be identified can be the sample from which
the polynucleotide is derived. A barcode can, for example, comprise
a nucleic acid sequence that when joined to a target polynucleotide
can serve as an identifier of the sample from which the target
polynucleotide was derived. In some embodiments, barcodes are at
least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more
nucleotides in length. In some embodiments, barcodes are shorter
than 10, 9, 8, 7, 6, 5, or 4 nucleotides in length. In some
embodiments, each barcode in a plurality of barcodes differ from
every other barcode in the plurality at at least three nucleotide
positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, or more
positions. In some embodiments, barcodes associated with some
polynucleotides are of different length than barcodes associated
with other polynucleotides. Barcodes can be of sufficient length
and comprise sequences that are sufficiently different to allow the
identification of samples based on barcodes with which they are
associated. In some embodiments, both the forward and reverse
adapter can comprise at least one of a plurality of barcode
sequences. In some embodiments, the first and second adaptor
comprises at least one of a plurality of barcode sequences. In some
embodiments, each reverse adapter comprises at least one of a
plurality of barcode sequences, wherein each barcode sequence of
the plurality of barcode sequences differs from every other barcode
sequence in the plurality of barcode sequences. In some
embodiments, both the first adapter and the second adapter comprise
at least one of a plurality of barcode sequences. In some
embodiments, barcodes for second adapter oligonucleotides are
selected independently from barcodes for first adapter
oligonucleotides. In some embodiments, first adapter
oligonucleotides and second adapter oligonucleotides having
barcodes are paired, such that adapters of the pair comprise the
same or different one or more barcodes. In some embodiments, the
methods of the invention further comprise identifying the sample
from which a target polynucleotide can be derived based on the
barcode sequence to which the target polynucleotide is joined. A
barcode can, for example, comprise a nucleic acid sequence that
when joined to a target polynucleotide serves as an identifier of
the sample from which the target polynucleotide was derived.
[0050] Appending of an adaptor(s) at the desired end of the
sequence region(s) of interest utilizing ligation can be suitable
for carrying out the disclosed methods. Various ligation modalities
are envisioned, dependent on the choice of nucleic acid, nucleic
acid modifying enzymes and the resulting ligatable end of the
nucleic acid. For example, when a blunt end product comprising the
target region/sequence of interest can be generated, blunt end
ligation can be suitable. Alternatively, where the cleavage can be
carried out using a restriction enzyme of known sequence
specificity, leading to the generation of cleavage sites with known
sequence overhangs, suitable ends of the adaptors can be designed
to enable hybridization of the adaptor to the cleavage site of the
sequence region of interest and subsequent ligation. Ligation also
can refer to any joining of two nucleic acid molecules that results
in a single nucleic acid sequences that can be further modified to
obtain the sequence of the nucleic acids in question. Reagents and
methods for efficient and rapid ligation of adaptors are
commercially available and are known in the art.
[0051] As used herein, the terms "amplifying", "amplification" and
to "amplify" a specific nucleic acid as used herein, can refer to a
procedure wherein multiple copies of the nucleic acid sample of
interest are generated, for example, in the form of DNA copies.
Many methods and protocols are known in the art to amplify nucleic
acids, such as e.g., PCR and qPCR.
[0052] As used herein, the term "cDNA" as used herein, can refer to
complementary DNA. The DNA can be synthesized in a reaction
catalyzed by the enzymes reverse transcriptase and DNA polymerase
from a messenger RNA (mRNA) template.
[0053] As used herein, the term "complementary" as used herein, can
refer to complementarity to all or only to a portion of a sequence.
The number of nucleotides in the hybridizable sequence of a
specific oligonucleotide primer or probe can be such that
stringency conditions used to hybridize the oligonucleotide primer
or probe can prevent excessive random non-specific hybridization.
The number of nucleotides in the hybridizing portion of the
oligonucleotide primer or probe can be at least as great as the
defined sequence on the target polynucleotide that the
oligonucleotide primer or probe hybridizes to, namely, at least 5,
at least 6, at least 7, at least 8, at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least about 20, and can be from about 6 to about 10 or 6 to about
12 of 12 to about 200 nucleotides, usually about 20 to about 50
nucleotides. The target polynucleotide/oligonucleotide can be
larger than the oligonucleotide primer, primers or probe.
[0054] As used herein, the term "denaturing" as used herein, can
refer to the separation of double stranded nucleic acid into single
strands. Denaturation can be achieved using any of the methods
known in the art including, but not limited to, physical, thermal,
and/or chemical denaturation.
[0055] As used herein, the acronym "FFPE" as used herein denotes
Formalin-Fixed, Paraffin Embedded. FFPE is a method used in
preservation of a tissue sample in which the sample can be fixed in
a formalin solution coupled with application of a wax referred to
as paraffin.
[0056] As used herein, the phrase "genomic DNA" as used herein, can
refer to chromosomal DNA, abbreviated as gDNA for genomic
deoxyribonucleic acid. gDNA includes the genetic material of an
organism.
[0057] As used herein, the term "genome" as used herein, can refer
to sequences, either DNA, RNA or cDNA derived from a patient, a
tissue, an organ, a single cell, a tumor, a specimen of an organic
fluid taken from a patient, freely circulating nucleic acid, a
fungus, a prokaryotic organism and a virus. A "transcriptome" as
used herein, can be all RNA sequences that can reflect a partial or
entire expressed genome of an organism.
[0058] As used herein, the term "kit" can refer to any system for
delivering materials. In the context of reaction assays, such
delivery systems can include elements allowing the storage,
transport, or delivery of reaction components such as
oligonucleotides, buffering components, additives, reaction
enhancers, enzymes and the like in the appropriate containers from
one location to another commonly provided with written instructions
for performing the assay. Kits can include one or more enclosures
or boxes containing the relevant reaction reagents and supporting
materials. The kit can comprise two or more separate containers
wherein each of those containers includes a portion of the total
kit components. The containers can be delivered to the intended
recipient together or separately.
[0059] As used herein, the phrase "nucleic acid (NA)-modifying
enzyme" as used herein, can refer to a DNA-specific modifying
enzyme. The NA-modifying enzyme can be selected for specificity for
double-stranded DNA. The enzyme can be a duplex-specific
endonuclease, a blunt-end frequent cutter restriction enzyme, or
other restriction enzyme. Examples of blunt-end cutters can include
Dral or Smal. The NA-modifying enzyme can be an enzyme provided by
NEW ENGLAND BIOLABS. The NA-modifying enzyme can be a homing
endonuclease (a homing endonuclease can be an endonuclease that
does not have a stringently-defined recognition sequence). The
NA-modifying enzyme can be a nicking endonuclease (a nicking
endonuclease can be an endonuclease that can cleave only one strand
of DNA in a double-stranded DNA substrate). The NA-modifying enzyme
can be a high fidelity endonuclease (a high fidelity endonuclease
can be an engineered endonuclease that has less "star activity"
than the wild-type version of the endonuclease). In some
embodiments, the NA-modifying enzyme can be a sequence and
duplex-specific, DNA modifying enzyme.
[0060] As used herein, the phrases "nucleic acid fragment" and
"specific nucleic acid" are used interchangeably and as used
herein, can refer to a portion of a nucleic acid sample. The
nucleic acids in the input sample can be fragmented into a
population of fragmented nucleic acid molecules or to
polynucleotides of one or more specific size range(s). The
fragments can have an average length from about 10 to about 10,000
nucleotides, from about 50 to about 2,000 nucleotides, from about
100-2,500, 10-1,000, 10-800, 10-500, 50-500, 50-250, or 50-150
nucleotides in length. The fragments can have an average length
less than 10,000 nucleotide, less than 5,000 nucleotides, less than
2,500 nucleotides, less than 2,000 nucleotides, less than 1,000
nucleotides, less than 500 nucleotides, such as less than 400
nucleotides, less than 300 nucleotides, less than 200 nucleotides,
or less than 150 nucleotides.
[0061] As used herein, the phrase "specific nucleic acid sequence"
or "specific sequence" as used herein, can be a polynucleotide
sequence of interest, for which digital measurement and/or
quantitation is desired, including but not limited to a nucleic
acid fragment. The specific sequence can be known or not known, in
terms of its actual sequence. A "template", as used herein, can be
a polynucleotide that contains the specific nucleic acid sequence.
The terms "specific sequence," "specific nucleic acid sequence,"
"specific nucleotide sequence," "regions of interest," or "sequence
of interest" and, variations thereof, are used interchangeably.
[0062] As used herein, the phrases "qualified nucleic acid" and
"qualifies the target nucleic acid fragment" as used herein, can
refer to a fragment of a gDNA or RNA sequence that is: i.) an
acceptable template for a DNA polymerase, i.e. the template can be
free of cross-links or inhibitors to the DNA polymerase, or ii.)
the template has a modification including, but not limited to,
attachment at the 5' and/or 3' end a polynucleotide sequence at
least one of a barcode, an adaptor, a sequence complementary to a
primer and so on such that the fragment can be modified for
purposes of quantitation, amplification, detection or to other
methods known to one of skill in the art of gDNA and cDNA sequence
analyses. The presence of inhibitors can be the result of using
gDNA obtained from a tissue sample that had undergone fixation in a
FFPE preparation.
[0063] As used herein, the term "oligonucleotide" can refer to a
polynucleotide chain, less than 200 residues long, e.g., between 15
and 100 nucleotides long, but can also encompass longer
polynucleotide chains. Oligonucleotides can be single-or
double-stranded. As used in this invention, the term
"oligonucleotide" can be used interchangeably with the terms
"primer", "probe" and "adaptor".
[0064] "PCR" is an abbreviation of term "polymerase chain
reaction," the nucleic acids amplification technology used in all
methods of the present invention, and which was originally
discovered and described by Mullis K. B. et al, U.S. Pat. No.
4,683,195 and Mullis K. B., U.S. Pat. No. 4,683,202. In some
embodiments, PCR employs two oligonucleotide primers for each
strand that are designed such as extension of one primer provides a
template for another primer in the next PCR cycle. Either one of a
pair of oligonucleotide primers can be named herein as a "forward"
or "reverse" primer with the purpose of distinguishing the
oligonucleotide primers in discussion. A PCR can consist of
repetition (or cycles) of (i) a denaturation step which separates
the strands of a double stranded nucleic acid, followed by (ii) an
annealing step, which allows primers to anneal to positions
flanking a sequence of interest; and then (iii) an extension step
which extends the primers in a 5' to 3' direction thereby forming a
nucleic acid fragment complementary to the target sequence. Each of
the above steps can be conducted at a different temperature using
an automated thermocycler. The PCR cycles can be repeated as often
as desired resulting in an exponential accumulation of a target DNA
fragment whose termini are usually defined by the 5' ends of the
primers used. Certain exceptions to this rule can apply, including
those described herein. Particular temperatures, incubation time at
each step and rates of change between steps depend on many factors
well-known to those of ordinary skill in the Art and the examples
can be found in numerous published protocols, for example,
McPherson M. J. et al. (1991 and 1995) and the like. Although
conditions of PCR can vary in a broad range, a double-stranded
target nucleic acid can be denatured at temperature >90.degree.
C., primers can be annealed at a temperature in the range
50-75.degree. C., and the extension can be performed in the range
72-78.degree. C.
[0065] The phrase "quantitative PCR" or "qPCR", as used herein, can
refer to a PCR designed to measure the abundance of one or more
specific target sequences in a sample. Quantitative measurements
can be made using one or more reference nucleic acid sequences that
can be assayed separately or together with a target nucleic acid.
Techniques for quantitative PCR are well known in the art and they
are exemplified in the following manuscripts that are incorporated
herein by reference: Gu Z. et al (2003) J. Clin. Microbiol.,
41:4636-4641; Becker-Andre M. and Hahlbrock K. (1989) Nucleic Acids
Res., 17:9437-9446; Freeman W. M. et al (1999) Biotechniques,
26:112-122, 124-125; Lutfalla G. and Uze G. (2006) Methods
Enzymol., 410:386-400; Clementi M. et al (1993) PCR Methods Appl.
2:191-196; Diviacco S. et al (1992) Gene, 122:313-320.
[0066] The term "portion", as used herein, can refer to less than
the total length of a nucleic acid sequence, a nucleic acid
sequence fragment, a specific nucleic acid sequence, a specific
nucleic acid fragment, a probe, a primer and the like. A portion
can be less than about 50 to about 2,000 nucleotides, from about
100-2,500, 10-1,000, 10-800, 10-500, 20-250, or 20-150 nucleotides
in length.
[0067] The term "primer", as used herein, can refer to an
oligonucleotide, generally with a free 3' hydroxyl group, that can
be capable of hybridizing or annealing with a template (such as a
specific polynucleotide, target DNA, target RNA, a primer extension
product or a probe extension product) and can be also capable of
promoting polymerization of a polynucleotide complementary to the
template. A primer can contain a non-hybridizing sequence that
constitutes a tail of the primer. A primer can still be hybridizing
to a target even though its sequences are not fully complementary
to the target.
[0068] The primers utilized herein can be oligonucleotides that are
employed in an extension reaction by a polymerase along a
polynucleotide template, such as in PCR, qPCR, an extension
reaction and the like. The oligonucleotide primer can be a
synthetic polynucleotide that can be single stranded, containing a
sequence at its 3'-end that can be capable of hybridizing with a
sequence of the target polynucleotide.
[0069] The 3' region of the primer that hybridizes with the
specific nucleic acid can comprise at least 80%, preferably 90%,
more preferably 95%, most preferably 100%, complementarity to a
sequence or to a primer binding site.
[0070] The term, "tail sequence" can refer to a non-hybridizing
sequence adjacent to and 5' of a primer or probe sequence. The term
"probe extension product" can refer to a DNA fragment resulting
from the hybridization of a probe and template directed synthesis
initiated from the probe, e.g., within a specific nucleic acid
sequence. The probe can be extended by a polymerase into an adaptor
sequence, if present and appended to the specific nucleic acid. The
resulting probe extension product can have both a first adaptor,
e.g., the adaptor appended to the specific nucleic acid sequence
and a second adaptor, e.g., found within the tail sequence of the
primer or probe.
[0071] A "random primer," as used herein, can be a primer that
comprises a sequence that can be designed not necessarily based on
a particular or to a specific sequence in a sample, but rather can
be based on a statistical expectation (or an empirical observation)
that the sequence of the random primer can be hybridizable (under a
given set of conditions) to one or more sequences in the sample. A
random primer can be an oligonucleotide or to a population of
oligonucleotides comprising a random sequence(s) in which the
nucleotides at a given position on the oligonucleotide can be any
of the four nucleotides, or any of a selected group of the four
nucleotides (for example only three of the four nucleotides, or
only two of the four nucleotides). As used herein, the notation
"n-random oligonucleotide" can refer to at least zero, at least
one, at least two, at least three, at least four, at least six, at
least eight, at least nine, at least 10 and so on, bases within an
adaptor or a priming site.
[0072] A "random nucleotide" and "n-random nucleotide sequence," as
used herein, can be a nucleotide that can comprise a sequence
within an adaptor or primer that can be designed not necessarily
based on a particular or to a specific sequence in a sample, but
rather can be based on a statistical expectation (or an empirical
observation) that the adaptor or primer having the random
nucleotide can be hybridizable (under a given set of conditions) to
one or more sequences in a primer, an adapter or a sample. A random
oligonucleotide can be an oligonucleotide or a population of
oligonucleotides comprising a random sequence(s) in which the
nucleotides at a given position on the oligonucleotide can be any
of the four nucleotides, or any of a selected group of the four
nucleotides (for example only three of the four nucleotides, or
only two of the four nucleotides or only one of the nucleotides).
As used herein, the notation "n-random oligonucleotide" can refer
to at least zero, at least one, at least two, at least three, at
least four, at least six, at least seven, at least eight, at least
nine, at least 10 and so on, bases within an adaptor or a
primer.
[0073] The term, "sample" as used herein, can refer to any
substance containing or presumed to contain a nucleic acid of
interest, and thus includes a sample of nucleic acid, cells,
organisms, tissue, fluids (e.g., spinal fluid or lymph fluids),
organic fluid taken from a patient, and sample including but not
limited to blood, plasma, serum, urine, tears, stool, respiratory
and genitourinary tracts, saliva, fragments of different organs,
tissue, blood cells, circulating tumor cell (CTC) or a disseminated
tumor cell (CTD), bone, samples of in vitro cell cultures or
specimens that have been suspected to contain nucleic acid
molecules.
[0074] The phrase, "communicable infection," and "communicable
disease," can refer to infections and diseases transmittable from
person to person; animal-to-animal, animal to human, or human to
animal direct contact or incidental contact by virtue of
proximity.
[0075] The term "PCR duplicate", as used herein, can refer to any
sequencing read that is derived from the same original nucleic acid
molecule and so, the same primer/probe extension product sequence,
as another sequencing read and is therefore not representative of a
unique nucleic acid molecule.
[0076] The term "probe", as used herein, can refer to an
oligonucleotide sequence. The probe can be complementary to a probe
target region. The probe sequence complementary to the probe target
region can be less than about 200 residues long, between about 15
and 100 nucleotides long, but can also be intended to encompass
longer polynucleotide chains. Probe target regions can be single-or
double-stranded. The probe target region provides a hybridization
site for a complementary probe that undergoes extension using a
polymerase.
[0077] The term "probe target region", as used herein, can refer to
a region within a genomic or transcriptomic database or within a
genome or transcriptome sequence to which a probe has been
designed. The region may extend beyond the specific complementary
region and include flanking regions of the genome or transcriptome.
The aligned probe sequence to its probe target region can provide
verification of the specificity of probe annealing and so too the
probe extension product and thus the specific nucleic acid molecule
being counted.
[0078] The probe target region is within a specific nucleic acid
sequence. The probe target region can be about 500 residues long
and can also be between about 80 and 1000 residues. As used herein,
the term "probe target region" can be used interchangeably with the
term "probe hybridization site" and "probe annealing site".
[0079] The term "verified probe" or "verified probe sequence", as
used herein, can refer to the sequence of the probe that has been
verified to be present and hybridized to the intended specific
target nucleic acid from the resulting sequencing data.
[0080] Reference will now be made in detail to exemplary
embodiments of the disclosed invention. While the disclosed methods
and compositions will be described in conjunction with the
exemplary embodiments, it will be understood that these exemplary
embodiments are not intended to limit the disclosed invention. On
the contrary, the disclosed invention is intended to encompass
alternatives, modifications and equivalents, which can be included
in the spirit and scope of the disclosed invention.
[0081] In some embodiments, disclosed herein are methods and
compositions for the quantitation of specific nucleic acid
sequences of interest from a sample comprising a plurality of
nucleic acids. The methods described herein can amplify specific
nucleic acid sequences using a conventional adaptor, sequence
specific probe target region probes, polymerase and ligation
enzymes and ligation. The methods can further enable digital
measurement of at least a first specific nucleic acid sequence
derived from a transcriptome or genomic DNA.
[0082] Digital gene expression has been performed multiple ways,
with each having significant drawbacks, thus making a new
methodology important for performing proper digital counting of
nucleic acid molecules. The current methods for digital nucleic
acid counting can include digital PCR, high throughput sequencing
and hybridization based counting as performed by the Nanostring
n-counter system.
[0083] Digital PCR can be performed by diluting the starting
nucleic acid material to the point of obtaining one copy per PCR
vessel, either in a well in a plate or an emulsion droplet. End
Point PCR can be performed for a given set of target primers and
the number of wells or droplets that are positive for an
amplification event can be counted. The main drawbacks to this
method are the problem of obtaining exactly one copy of target
nucleic acid per vessel based on the Poisson distribution, and also
the reaction can be very limited to a small number of targets per
nucleic acid sample that can be interrogated (low multiplex
capability).
[0084] The n-counter system of Nanostring utilizes a probe
hybridization scheme with single molecule resolution to count input
nucleic acids by measuring fluorescent signals. The major drawbacks
to this technology are the low multiplexing, due to the fluorescent
tags that must be used, and the inability to target different
regions on the same molecule. For example, due to the size of the
fluorescent tags used, the n-counter system can be unable to
interrogate the presence of two exons within the same RNA
transcript.
[0085] High throughput sequencing can be considered an excellent
method for digital counting of nucleic acid molecules, but it too
suffers from major drawbacks. For both genomic DNA as well as RNA
counting, the nucleic acids can be randomly sheared prior to
sequencing. This random shearing can introduce bias into the base
composition of the target, resulting in uneven amplification or
sequencing of a given target of interest. The major source of
ambiguity in counting nucleic acid fragments can be based on the
methods currently use to count. That is, for a given gene of
interest (or genomic target region), the number of sequencing reads
obtained must be normalized by the size of the target region so
that targets of different sizes, which would therefore necessarily
generate different numbers of sequencing reads, can be compared to
each other. The ambiguity occurs because the size of a target
region is not necessarily fixed between samples since different
length isoforms of the same gene exist at varying abundances. This
can be most easily seen in the case of RNA sequencing, but applies
equally to genomic DNA.
[0086] In RNA sequencing, gene counts can be expressed as RPKM or
FPKM (reads/fragments per thousand million or fragments per
thousand million) depending on the type of data generated. The
sequencing data counts can be determined by the number of reads (or
fragments in the case of paired end sequencing), the size of the
target RNA (in kilobases), and the number of total sequencing reads
(in millions). The problem lies in measuring the size of the target
RNA; one size is assumed across all samples. However, it is well
known that through alternative exon usage, the size of RNA can
differ by up to many kb of sequence between different samples, thus
potentially altering the size variable in the RPKM/FPKM measurement
between two samples. The changes in size measurement for one gene
additionally effect the RPKM/FPKM measurements for all genes in the
sample as for a fixed number of sequencing reads, altering the size
of one gene through alternative exon usage will change the number
of reads from other genes. Just as described with RNA sequencing,
genomic DNA counting can suffer from similar problems when taking
into account partial duplications and deletions, which alter the
size of the target region of interest between samples.
[0087] In some embodiments, disclosed herein are methods and
compositions for the digital measurement of specific nucleic acid
sequences from a sample having a plurality of nucleic acids. The
nucleic acids can be DNA, or RNA. The nucleic acids can be single
or double stranded. The DNA can be genomic DNA, cDNA, a DNA/RNA
hybrid or any combination thereof. In some embodiments, the nucleic
acids in an input sample can be double stranded DNA. In some
embodiments, the method includes fragmenting nucleic acids in an
input sample to generate nucleic acid fragments. In some
embodiments, the sample is not fragmented. In some embodiments,
fragmentation of the nucleic acids can be achieved through methods
known in the art or described herein for fragmenting nucleic acids
that can include, but are not limited to, physical (i.e.
sonication), and/or enzymatic (i.e. restriction enzyme treatment)
fragmentation reactions.
[0088] Physical fragmentation methods can include nebulization,
sonication, and/or hydrodynamic shearing. In some embodiments, the
fragmentation can be accomplished mechanically comprising
subjecting the nucleic acids in the input sample to acoustic
sonication. In some embodiments, the fragmentation comprises
treating the nucleic acids in the input sample with one or more
enzymes under conditions suitable for the one or more enzymes to
generate double-stranded nucleic acid breaks. Examples of enzymes
useful in the generation of nucleic acid or polynucleotide
fragments can include sequence specific and non-sequence specific
nucleases. Non-limiting examples of nucleases can include DNase I,
Fragmentase, restriction endonucleases, variants thereof, and
combinations thereof. Reagents for carrying out enzymatic
fragmentation reactions are commercially available, for example as
provided by NEW ENGLAND BIOLABS. For example, digestion with DNase
I can induce random double-stranded breaks in DNA in the absence of
Mg++ and in the presence of Mn++. In some embodiments,
fragmentation comprises treating the nucleic acids in the input
sample with one or more restriction endonucleases. Fragmentation
can produce fragments having 5' overhangs, 3' overhangs, blunt
ends, or a combination thereof. In some embodiments, such as when
fragmentation comprises the use of one or more restriction
endonucleases, cleavage of sample polynucleotides leaves overhangs
having a predictable sequence.
[0089] In some embodiments, the nucleic acids in the input sample
can be fragmented into a population of fragmented nucleic acid
molecules or to polynucleotides of one or more specific size
range(s). In some embodiments, the fragments can have an average
length from about 10 to about 10,000 nucleotides. In some
embodiments, the fragments can have an average length from about 50
to about 2,000 nucleotides. In some embodiments, the fragments can
have an average length from about 100-2,500, 10-1,000, 10-800,
10-500, 50-500, 50-250, or 50-150 nucleotides. In some embodiments,
the fragments can have an average length less than 10,000
nucleotide, such as less than 5,000 nucleotides, less than 2,500
nucleotides, less than 2,500 nucleotides, less than 1,000
nucleotides, less than 500 nucleotides, such as less than 400
nucleotides, less than 300 nucleotides, less than 200 nucleotides,
or less than 150 nucleotides.
[0090] In some embodiments, fragmentation of the nucleic acids can
be followed by end repair of the nucleic acid fragments. In some
embodiments, non-fragmented samples can undergo end repair. End
repair can include the generation of blunt ends, non-blunt ends
(i.e. sticky or cohesive ends), or single base overhangs such as
the addition of a single dA nucleotide to the 3'end of the nucleic
acid fragments by a polymerase lacking 3'-exonuclease activity. End
repair can be performed using any number of enzymes and/or methods
known in the art including, but not limited to, commercially
available kits such as the ultralow next-generation sequencing
library system sold under the trademark OVATION Ultralow NGS
Library System by NuGEN. In some embodiments, end repair can be
performed on double stranded DNA fragments to produce blunt ends
wherein the double stranded DNA fragments contain 5' phosphates and
3' hydroxyls. In some embodiments, the double-stranded DNA
fragments can be blunt-end polished (or "end repaired") to produce
DNA fragments having blunt ends, prior to being joined to adapters.
Generation of the blunt ends on the double stranded fragments can
be generated by the use of a single strand specific DNA exonuclease
such as for example exonuclease 1, exonuclease 7 or a combination
thereof to degrade overhanging single stranded ends of the double
stranded products. Alternatively, the double stranded DNA fragments
can be blunt ended by the use of a single stranded specific DNA
endonuclease, for example, but not limited to, mung bean
endonuclease or SI endonuclease. Alternatively, the double stranded
products can be blunt ended by the use of a polymerase that
comprises single stranded exonuclease activity such as for example
T4 DNA polymerase, or any other polymerase comprising single
stranded exonuclease activity or a combination thereof to degrade
the overhanging single stranded ends of the double stranded
products. In some cases, the polymerase comprising single stranded
exonuclease activity can be incubated in a reaction mixture that
does or does not comprise one or more dNTPs. In other cases, a
combination of single stranded nucleic acid specific exonucleases
and one or more polymerases can be used to blunt end the double
stranded fragments generated by fragmenting the sample comprising
nucleic acids. In still other cases, the nucleic acid fragments can
be made blunt ended by filling in the overhanging single stranded
ends of the double stranded fragments. For example, the fragments
can be incubated with a polymerase such as T4 DNA polymerase or
Klenow polymerase or a combination thereof in the presence of one
or more dNTPs to fill in the single stranded portions of the double
stranded fragments. Alternatively, the double stranded DNA
fragments can be made blunt by a combination of a single stranded
overhang degradation reaction using exonucleases and/or
polymerases, and a fill-in reaction using one or more polymerases
in the presence of one or more dNTPs. Kits commercially available
for blunt end repair or end polishing also include blunting kits
sold under the trademark NEB and end repair kits sold under the
trademark NEBNext, each sold by NEW ENGLAND BIOLABS.
[0091] In some embodiments the fragmented specific nucleic acid can
be denatured into single-stranded nucleic acid fragments. In some
embodiments, the non-fragmented sample can be denatured into
single-stranded nucleic acid strands. Methods for denaturing
double-stranded nucleic acid into single-stranded nucleic acid are
well known to one of skill in the art. Methods include but are not
limited to heat denaturation, chemical denaturation and the
like.
[0092] The methods described herein for quantitating specific
nucleic acid fragment sequences or non-fragmented nucleic acid
sample sequences can further include appending at least a first
adaptor to the nucleic acid fragments or non-fragmented nucleic
acid sample sequences generated by the methods described herein. In
some embodiments, the at least first adaptor can be a forward
adaptor. Appending the at least first adaptor to the nucleic acid
fragments or non-fragmented nucleic acid sample sequences generated
by methods described herein can be achieved using a ligation
reaction or a priming reaction. In some embodiments, appendage of
an at least first adaptor to the nucleic acid fragments or
non-fragmented nucleic acid sample sequences comprises ligation. In
some embodiments, ligation of the at least first adaptor to the
nucleic acid fragments or non-fragmented nucleic acid sample
sequences can be following end repair of the nucleic acid fragments
or non-fragmented nucleic acid sample sequences. In some
embodiments, the ligation of the at least first adaptor to the
nucleic acid fragments or non-fragmented nucleic acid sample
sequences can be following generation of the nucleic acid fragments
or non-fragmented nucleic acid sample sequences without end repair
of the nucleic acid fragments or non-fragmented nucleic acid sample
sequences.
[0093] The at least first adaptor can be any type of adaptor known
in the art including, but not limited to, conventional duplex or
double stranded adaptors in which the adaptor comprises two
complementary strands. In some embodiments, the first adaptor can
be a double stranded DNA adaptor. In some embodiments, the first
adaptor can be an oligonucleotide of known sequence and, thus,
allow generation and/or use of sequence specific primers for
amplification and/or sequencing of any polynucleotides to which the
at least first adaptor(s) can be appended or attached. In some
embodiments, the first adaptor can be a conventional duplex
adaptor, wherein the first adaptor comprises sequence well known in
the art. In some embodiments, the methods described herein can
involve the use of a first duplex adaptor comprising double
stranded DNA of known sequence that can be blunt ended and can be
coupled to the double stranded nucleic acid fragments generated by
the methods described herein in one orientation. In some
embodiments, a first adaptor can be appended or ligated to a
library of nucleic acid fragments generated by the methods
described herein such that each nucleic acid fragment in the
library of nucleic acid fragments or non-fragmented nucleic acid
sample in the library of non-fragmented nucleic acids comprises the
first adaptor ligated to one end. In some embodiments, the at least
first adaptor can be appended or ligated to a single-stranded
nucleic acid fragment or a non-fragmented nucleic acid sample
sequences and can be incorporated into a probe extension
product.
[0094] Ligation of the at least first adaptor to the nucleic acid
fragments or non-fragmented nucleic acid sample sequence generates
a first adaptor specific nucleic acid fragment complex or a first
adaptor non-fragmented nucleic acid sample sequence, a ligation
product. In some embodiments, the first adaptor specific nucleic
acid fragment complex can be denatured. In some embodiments, a
first adaptor non-fragmented nucleic acid sample sequence can be
denatured. Denaturation can be achieved using any of the methods
known in the art including, but not limited to, physical, thermal,
and/or chemical denaturation. In some embodiments, denaturation can
be achieved using thermal or heat denaturation. In some
embodiments, denaturation of the at least first adaptor specific
nucleic acid fragment complex or the at least first adaptor
non-fragmented nucleic acid sample sequence generates single
stranded nucleic acid fragments or non-fragmented nucleic acid
sample sequence comprising the at least first adaptor sequence at
only the 5' end of the nucleic acid fragments or non-fragmented
nucleic acid sample sequence as depicted, for example, in FIG.
1.
[0095] In some embodiments, the nucleic acid fragments or
non-fragmented nucleic acid sample sequences comprising first
adaptor sequence appended to either the 5' end or both the 5' and
3' end can be denatured to generate single stranded nucleic acid
fragments or non-fragmented nucleic acid sample sequence comprising
first adaptor sequence appended to either the 5' end or both the 5'
and 3' end. In some embodiments, the methods of the present
invention described herein can be used to generate a plurality of
single stranded nucleic acid fragments or non-fragmented nucleic
acid sample sequence comprising first adaptor sequence appended to
either the 5' end or both the 5' and 3' end. In some embodiments,
an oligonucleotide probe comprising at a first end sequence
complementary to a probe target region sequence of interest present
in a single stranded specific nucleic acid and at a second end
sequence from a second adaptor, wherein the second adaptor sequence
is not complementary to the probe target region can be annealed to
the single stranded specific nucleic acid fragments or
non-fragmented nucleic acid sample sequence. In some embodiments,
the second adaptor sequence can be sequence from a reverse
adaptor.
[0096] In some embodiments, the probe target region sequence of
interest can be present in one or more of the single stranded
specific nucleic acid fragments or non-fragmented nucleic acid
sample sequences. In some embodiments, different or distinct probe
target region sequences of interest can be present in one or more
of the single stranded nucleic acid fragments or non-fragmented
nucleic acid sample sequences. In some embodiments, one or more
oligonucleotides can comprise sequence complementary to the same
sequence of interest present in one or more single stranded nucleic
acid fragments or non-fragmented nucleic acid sample sequences. In
this embodiment, the one or more oligonucleotides can comprise
sequence that can be complementary to different parts or to regions
of the same sequence of interest. In some embodiments, the
different regions can be adjacent to each other. In some
embodiments, the different regions can be non-adjacent to each
other. In some embodiments, the one or more oligonucleotides that
comprise sequence complementary to the same target nucleic acid
sequence of interest can further comprise the same second adaptor
sequence. In some embodiments, one or more probe oligonucleotides
can comprise sequence complementary to different or to distinct
sequences of interest that can be present in one or more single
stranded nucleic acid fragments or non-fragmented nucleic acid
sample sequence. In some embodiments, the one or more
oligonucleotide probes that comprise sequence complementary to
different or to distinct target nucleic acid sequences of interest
and can further comprise the same second adaptor sequence. In some
embodiments, the sequence complementary to the target sequence of
interest can be at the 3' end of the oligonucleotide probe and the
second adaptor sequence can be at the 5' end of the
oligonucleotide. In some embodiments, the second adaptor sequence
can be non-complementary to the target nucleic acid sequence of
interest. In this manner, the second adaptor sequence serves as a
tail. The second adaptor sequence can be a conventional adaptor
sequence. In some embodiments, the second adaptor sequence can be a
conventional adaptor sequence that can be different than or
distinct from the sequence of the first adaptor appended to the
single stranded nucleic acid fragment or non-fragmented nucleic
acid sample sequence as described above. In some embodiments, the
second adaptor sequence can be of known sequence and, thus, allow
generation and/or use of sequence specific primers for
amplification and/or sequencing of any polynucleotides to which the
second adaptor sequence can be appended or attached. In a separate
embodiment, the oligonucleotide probe can be annealed to the
specific nucleic acid fragments or non-fragmented nucleic acid
sample sequences comprising the first adaptor sequence appended to
either the 5' end or both the 5' and 3' end without prior
denaturation. In this embodiment, annealing of the oligonucleotide
can be via formation of a triple helix or triplex between the
oligonucleotide and a double stranded nucleic acid fragment or
non-fragmented nucleic acid sample sequence comprising the first
adaptor sequence appended to either the 5' end or both the 5' and
3' ends of the double stranded nucleic acid fragment or
non-fragmented nucleic acid sample sequence. In this embodiment,
the double stranded nucleic acid fragment or non-fragmented nucleic
acid sample sequence comprises a sequence of interest and can be
present amongst a plurality of double stranded nucleic acid
fragments or non-fragmented nucleic acid sample sequence comprising
first adaptor sequence appended to either the 5' end or both the 5'
and 3' end. Further to this embodiment, the oligonucleotide probe
comprises sequence complementary to the probe target region in the
double stranded specific nucleic acid fragment or non-fragmented
nucleic acid sample sequence. Overall, the use of the
oligonucleotide probe comprising sequence complementary to a probe
target region sequence of interest present in a nucleic acid
fragment or non-fragmented nucleic acid sample sequence amongst one
or more or a plurality of specific nucleic acid fragments or
non-fragmented nucleic acid sample sequences allows for selective
binding and subsequent enrichment of said nucleic fragment or
non-fragmented nucleic acid sample sequence using the methods
described herein.
[0097] Following annealing of the oligonucleotide probe as
described above, a polymerase can be used to extend the
oligonucleotide probe. In some embodiments, the polymerase can be a
DNA dependent DNA polymerase. In some embodiments, the DNA
dependent DNA polymerase can be any of the DNA dependent DNA
polymerases as described herein and extension of the
oligonucleotide can be by any of the methods known in the art. In
some embodiments, an oligonucleotide probe comprising the second
adaptor sequence, wherein the second adaptor sequence is not
complementary to the probe target region nucleic acid, and sequence
complementary to a probe target region sequence of interest present
in a specific nucleic acid fragment comprising a first adaptor
appended to one and/or both ends can be annealed to the nucleic
acid fragment and extended with a polymerase to generate an probe
extension product comprising the first adaptor sequence at a first
end and the second adaptor sequence at a second end. In some
embodiments, the specific nucleic acid fragment can be present
amongst a plurality of nucleic acid fragments comprising first
adaptor appended to one and/or both ends. In this embodiment, the
probe extension product can only be generated for a nucleic acid
fragment that contains the probe target region sequence of
interest.
[0098] In some embodiments, the probe extension product generated
by the methods described herein can be subjected to an
amplification reaction. In some embodiments, the amplification
reaction can be exponential, and can be carried out at various
temperature cycles. The amplification reaction can be an isothermal
reaction. In some embodiments, the amplification can be a
quantitative polymerase chain reaction (qPCR). In some embodiments,
the amplification reaction can be isothermal. In some embodiments,
the probe extension product comprises at least first adaptor
sequence on one end and a second adaptor sequence on the other end
as generated by the methods described herein. In some embodiments,
the probe extension product can be amplified using a first primer
comprising sequence complementary to the first adaptor and a second
primer having sequence complementary to a 5' tail sequence, in the
strand complementary to the probe target region within the specific
nucleic acid strand. In this manner probe extension products
comprising both the first adaptor sequence and a probe target
region can be amplified and so enriched. Probe extension products
having both the at least first adaptor sequence and a probe target
region sequence are amplified, wherein an amplified probe extension
product generated from said ligated specific nucleic acid fragment
or non-fragmented nucleic acid sample sequence can be quantitated.
In some embodiments, the at least first adaptor sequence and/or the
second adaptor sequence can comprise an identifier sequence. In
some embodiments, the identifier sequence can be a barcode
sequence. In some embodiments, the barcode sequence can be unique
for the at least first adaptor. In some embodiments, the at least
first adaptor and/or the second adaptor sequence can comprise
sequence that can be used for downstream applications such as, for
example, but not limited to, sequencing and specific nucleic acid
identification after a sequencing reaction. In some embodiments,
the at least first adaptor and/or the second adaptor sequence can
comprise flow cell sequences 33 and 35 (FIG. 5) that can be used
for sequencing with the sequencing method developed by Illumina and
described herein.
[0099] A schematic of a disclosed embodiment of the methods
described herein for quantitating specific nucleic acid sequence
fragments of interest is illustrated in FIG. 1 and FIG. 2. The
numbering scheme used in the figures is illustrative only. The same
number appearing in more than one figure is not intended to
indicate an identical oligonucleotide sequence, in whole or in part
but rather a component, site or region of reference for practicing
the disclosed methods.
[0100] The methods of FIG. 1 and FIG. 2 illustrate generation of a
ligated library of nucleic acid fragments, non-fragmented nucleic
acid samples or inserts wherein each nucleic acid sequence of the
ligated library comprises a common forward read priming site within
the adaptor and a specific probe target region sequence such that
PCR amplification using a primer complementary to the forward read
priming site and a primer complementary to the reverse read priming
site within the probe extension product comprising the probe target
region provides sequencing coverage to allow quantitation of the
specific nucleic acid molecule having the specific probe target
region sequence.
[0101] FIG. 1 illustrates the use of sheared gDNA. Sheared DNA 8
has adaptor 11 ligated to the 5' end of gDNA having specific
nucleic acid fragment 10. The fragment 10 includes probe target
region 50. The adaptor can comprise at least one of a sequencing
read 1 forward oligonucleotide priming site 12, a n-random
oligonucleotide base(s) such as a 6N oligonucleotide sequence 14,
an index base oligonucleotide sequence 16, and depending on the
high throughput sequencing method used, an index priming site 18.
Upon ligation of the adaptor 11 the specific nucleic acid fragment
10 can have a unique identifier sequence label, the index read plus
the n-random oligonucleotide. The index sequence 16 is used to
identify the specific nucleic acid sample and the 6N
oligonucleotide sequence 14 is used in marking duplicate sequencing
reads. Probe oligonucleotide sequence 19 having a 5' tail
oligonucleotide sequence 20 can be complementary to and hybridize
to probe target region 50 and can be extended in a single primer
extension reaction in the presences of dNTPs and DNA polymerase
through the adaptor 11. The resulting probe extension product 22
can be amplified using forward primer 24 that can be partially
complementary to index priming site 18 and reverse primer 26 that
can be partially complementary to the reverse complement of the 5'
tail sequence 20. The amplification reaction enriches the presence
of specific nucleic acid 10 having probe target region 50 to
generate a library of specific nucleic acid sequences.
[0102] As illustrated in FIG. 2 a similar single primer extension
reaction can be applicable to cDNA. cDNA 7 has adaptor 11 ligated
to the 5' end of specific nucleic acid fragment 9. The fragment 9
includes probe target region 60. The adaptor can comprise at least
one of a forward sequencing read oligonucleotide priming site 12, a
known random oligonucleotide base(s) such as a 6N oligonucleotide
sequence 14, an index base oligonucleotide sequence 16, and
depending on the high throughput sequencing method used, an index
sequencing read priming site 18. The index sequence 16 is used to
identify the specific nucleic acid sample and the 6N
oligonucleotide sequence 14 is used in identifying duplicate
sequencing reads. Probe oligonucleotide sequence 19 having a 5'
tail oligonucleotide sequence 20 can be complementary to and
hybridizes to probe target region sequence 60 and can be extended
in a single primer extension reaction in the presences of dNTPs and
DNA polymerase through the adaptor 15. The resulting probe
extension product 21 can be amplified using forward primer 24 that
can be partially complementary to 18 and reverse primer 26 that can
be partially complementary to the reverse complement of the 5' tail
sequence 20. The amplification reaction enriches the presence of
specific nucleic acid 9 having probe target region 60 to generate a
library of specific nucleic acid sequences.
[0103] A schematic of a disclosed embodiment of the methods
described herein for quantitating specific nucleic acid sequence
fragments of interest is illustrated in FIG. 3 and FIG. 4 for
double stranded gDNA. The numbering scheme used in the figures is
illustrative only. The same number appearing in more than one
figure is not intended to indicate an identical oligonucleotide
sequence, in whole or in part but rather a component, site or
region of reference for practicing the disclosed methods.
[0104] The methods of FIG. 3 and FIG. 4 illustrate generation of a
sequencing library of nucleic acid fragments, non-fragmented
nucleic acid samples or inserts wherein each nucleic acid sequence
of the sequencing library comprises a common forward priming site
within one adaptor and a specific probe target region sequence such
that there can be sequencing coverage to allow quantitation of the
specific nucleic acid molecule having the specific probe target
region sequence. The sequencing can be done using a sequencing
library made from the ligated probe extension products with or
without PCR amplification using a primer complementary to the
common forward priming site and a primer complementary to the
specific probe target region sequence within the specific nucleic
acid sequence.
[0105] FIG. 3 illustrates the use of sheared gDNA. Sheared gDNA
having specific nucleic acid 10 includes probe target region 50.
Probe oligonucleotide sequence 19, having a 5' tail oligonucleotide
sequence 20, can be complementary to and hybridizes to probe target
region sequence 50 and can be extended in a single probe extension
reaction in the presences of dNTPs and DNA polymerase through the
end of specific nucleic acid 10 creating double-stranded DNA. The
resulting probe extension product can have an adaptor ligated to
the 3' end of specific nucleic acid fragment 10. The adaptor can
comprise at least one of a forward sequencing read 1
oligonucleotide priming site 12, a n-random oligonucleotide base(s)
such as a 6N oligonucleotide sequence 14, an index base
oligonucleotide sequence 16, and depending on the high throughput
sequencing method used, an index priming site 18. The index
sequence 16 is used to identify the specific nucleic acid sample
and the 6N oligonucleotide sequence 14 is used in marking duplicate
sequencing reads. The ligated product 22 can be amplified using
forward primer 24 that can be partially complementary to index
priming site 18 and reverse primer 26 that can be partially
complementary to the reverse complement of 5' tail sequence 21. The
amplification reaction can enrich for the presence of specific
nucleic acid 10 having probe target region 50 to generate a library
of specific nucleic acid sequences.
[0106] FIG. 4 illustrates the use of sheared gDNA. Sheared gDNA
having specific nucleic acid 10 includes probe target region 50.
Probe oligonucleotide sequence 19, having a 5' tail oligonucleotide
sequence 20, can be complementary to and hybridizes to probe target
region sequence 50 and can be extended in a single probe extension
reaction in the presences of dNTPs and DNA polymerase through the
end of gDNA 10 creating double-stranded DNA. The resulting probe
extension product can be digested by a restriction enzyme 70.
Exemplary restriction enzymes include but are not limited to Xbal,
EcoRI, EcoRV, and BamHI. Following restriction enzyme digestion an
adaptor can be ligated to the end of double-stranded gDNA having
specific nucleic acid fragment 10. The adaptor can comprise at
least one of a Read 1 forward oligonucleotide priming site 12, a
n-random oligonucleotide base(s) such as a 6N oligonucleotide
sequence 14, an index base oligonucleotide sequence 16, and
depending on the high throughput sequencing method used, an index
priming site 18. The index sequence 16 is used to identify the
specific nucleic acid sample and the 6N oligonucleotide sequence 14
is used in marking duplicate sequencing reads. The ligated product
22 can be amplified using forward primer 24 that can be partially
complementary to index priming site 18 and reverse primer 26 that
can be partially complementary to 5' tail sequence 21 as
illustrated in FIG. 3. The amplification reaction can enrich for
the presence of specific nucleic acid 10 having probe target region
50 to generate a library of specific nucleic acid sequences.
[0107] As illustrated in FIG. 5 (numbering refers the numbering
used in FIG. 1 or FIG. 2) a similar single primer extension
reaction can be applicable to either gDNA or cDNA to create a
sequencing library for a variety of sequencing platforms. The gDNA
or cDNA (sheared or not) 10 or 9 has adaptor 11 ligated to the 5'
end of specific nucleic acid fragment 10 or 9. The fragment 10 or 9
includes probe target region 50 or 60, respectively. The adaptor
can comprise at least one of a forward oligonucleotide priming site
12, a known random oligonucleotide base(s) such as a 6N
oligonucleotide sequence 14, an index base oligonucleotide sequence
16, and depending on the high throughput sequencing method used, an
index priming site 18. Probe oligonucleotide sequence 19 having a
5' tail oligonucleotide sequence 20 and can be complementary to and
hybridizes to probe target region sequence 50 or 60 and can be
extended in a single primer extension reaction in the presences of
dNTPs and DNA polymerase through the adaptor 11. The resulting
probe extension product 21 or 22 can be amplified using forward
primer 24 that can be partially complementary to 18 and reverse
primer 26 that can be partially complementary to 5' tail sequence
20. The amplification reaction enriches the presence of specific
nucleic acid 10 or 9 having probe target region 50 or 60 to
generate a library of specific nucleic acid sequences.
[0108] Libraries can be prepared using the target enrichment
systems sold under the trademark OVATION by NuGEN by selectively
amplifying by PCR those probe extension product sequences having
the selected probe target region sequence of interest. FIG. 5
illustrates an example of a nucleic acid library used in high
throughput sequencing when using the Illumina high throughput
sequencing platform. Specific sequence read regions of each
sequence library can be analyzed for digital measurement of e.g.
gene expression or copy number variation quantitation.
[0109] In some embodiments, the specific nucleic acids can be
tagged with an indicator molecule, including but not limited to,
biotin. The tagged specific nucleic acid molecules can then be
distinguished as originating from original sample molecules. In
some embodiments, attachment of an indicator molecule can be
accomplished via ligation or polymerase addition of a labeled
nucleotide, e.g., a biotinylated nucleotide. Probes that can be
complementary to a probe target region can then be hybridized to
the tagged nucleic acids with or without probe extension by
polymerase. In some embodiments, non-hybridizing probes are
removed, for example, by capturing the tagged nucleic acids via a
biotin/streptavidin interaction. In some embodiments, probes that
hybridized to the targets are captured along with the targets.
Following removal of non-hybridizes probes, the captured probes are
eluted off of the target nucleic acids and counted. In some
embodiments, counting can be done by sequencing via the Illumina
platform and counting those tags. In some embodiments, the probe
can be tagged with a nanopore or fluorescent tagging as is known to
one of skill in the art.
Input Nucleic Acid
[0110] The input can be a human nucleic acid. In some embodiments,
the input can be DNA. In some embodiments, the input human nucleic
acid can be complex DNA, such as double-stranded DNA, genomic DNA
or mixed DNA from more than one organism. In some embodiments, the
input can be RNA. In some embodiments, the RNA can be obtained and
purified using standard techniques in the art and can include RNAs
in purified or unpurified form, which can include, but are not
limited to, mRNAs, tRNAs, snRNAs, rRNAs, small non-coding RNAs,
microRNAs, polysomal RNAs, pre-mRNAs, intronic RNA, cell free RNA
and fragments thereof. The non-coding RNA, or ncRNA can include
snoRNAs, microRNAs, siRNAs, piRNAs and long ncRNAs. In some
embodiments, the DNA fragments can be derived from RNA that has
been converted to cDNA through a first strand synthesis reaction
using any of the methods well known in the art for generating cDNA
from an RNA template which can include, but is not limited to,
combining the RNA with a primer (i.e. random primer), and reverse
transcribing the RNA template with an RNA-dependent DNA polymerase.
In some embodiments, the DNA fragments can be derived from RNA that
has been converted to double stranded cDNA through a first and
second strand synthesis reaction using any of the methods well
known in the art.
[0111] In some embodiments, the input DNA can be cDNA made from a
mixture of genomes of different species. The input complex also can
be from a mixture of genomes of different humans. The input DNA can
be cDNA made from a mixture of genomes of different humans. The
input DNA can be of a specific species, for example, human, rat,
mouse, other animals, specific plants, bacteria, algae, viruses,
and the like. The input complex also can be from a mixture of
genomes of different species such as host-pathogen, bacterial
populations and the like. Alternatively, the input nucleic acid can
be from a synthetic source. The input DNA can be mitochondrial DNA.
The input DNA can be cell-free DNA. The cell-free DNA can be
obtained from, e.g., a serum or plasma sample. The input DNA can
comprise one or more chromosomes. For example, in cases wherein the
input DNA can be from a human, the DNA can comprise one or more of
chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, X, or Y. The DNA can be from a linear or
circular genome. The DNA can be plasmid DNA, cosmid DNA, bacterial
artificial chromosome (BAG), or yeast artificial chromosome (YAC).
The input DNA can be from more than one individual human. The input
DNA can be double stranded or single stranded. The input DNA can be
part of chromatin. The input DNA can be associated with
histones.
[0112] In some embodiments, the probe oligonucleotide can be
directed to a specific nucleic acid sequence of interest and can be
designed to hybridize to single-stranded specific nucleic acid
targets having a probe target region within the specific nucleic
acid. In some embodiments, the probes targeting the selected
sequence regions of interest can be designed to hybridize to
single-stranded DNA or cDNA probe target regions. In the case where
the input nucleic acid sample comprises genomic DNA or other
double-stranded DNA, the input nucleic acid sample can be first
denatured to render the target single stranded and enable
hybridization of the oligonucleotide probes to the desired probe
target region sequence regions of interest. In some embodiments,
the other double-stranded DNA can be double-stranded cDNA generated
by first and second strand synthesis of one or more target RNAs. In
these embodiments, the methods and compositions described herein
can allow for region-specific enrichment and amplification of a
plurality of specific nucleic acid sequence regions of interest
containing a plurality of probe target regions. In some
embodiments, the methods and compositions described herein allow
for multiplex amplification, enrichment and quantitation of at
least two or more distinct specific nucleic acid sequence fragments
or non-fragmented nucleic acid sample sequences, each having a
distinct region of interest containing a corresponding distinct
probe target region.
[0113] In other embodiments, the probes targeting the selected
sequence regions of interest can be designed to hybridize to
double-stranded nucleic acid target fragments or non-fragmented
nucleic acid sample sequences, without denaturation of the double
stranded nucleic acids fragment or non-fragmented nucleic acid
sample sequence. In other embodiments, the probes targeting the
selected sequence regions of interest can be designed to hybridize
to a double-stranded DNA target, without denaturation of the dsDNA.
In these embodiments, the probes targeting the selected sequence
regions of interest can be designed to form a triple helix
(triplex) at the selected sequence regions of interest. The
hybridization of the probes to the double-stranded DNA sequence
regions of interest can be carried out without prior denaturation
of the double stranded nucleic acid sample. In such embodiments,
the methods and compositions described herein can allow for
region-specific quantitation as well as strand-specific
amplification and quantitation of sequence regions of interest.
This method can be useful for generation of copies of strand
specific sequence regions of interest from complex nucleic acid
without the need to denature the dsDNA input DNA, thus enabling
quantitation and analysis of multiplicity of sequence regions of
interest in the native complex nucleic acid sample. The method can
find use for studies and analyses carried out in situ, enable
studies and analysis of complex genomic DNA in single cells or
collection of very small well-defined cell population, as well as
permit the analysis of complex genomic DNA without disruption of
chromatin structures.
[0114] In some embodiments, disclosed herein are adaptors
comprising an additional identifier sequence, e.g. a barcode
sequence. In some embodiments, the at least first adaptor comprises
at least one of a plurality of barcode sequences. In some
embodiments, each reverse adapter comprises at least one of a
plurality of barcode sequences, wherein each barcode sequence of
the plurality of barcode sequences differs from every other barcode
sequence in the plurality of barcode sequences. In some
embodiments, barcodes for second adapter oligonucleotides can be
selected independently from barcodes for at least first adapter
oligonucleotides. In some embodiments, first adapter
oligonucleotides and second adapter oligonucleotides having
barcodes can be paired, such that adapters of the pair comprise the
same or different one or more barcodes. In some embodiments, the
methods of the invention can further comprise identifying the
sample from which a target polynucleotide is derived based on the
barcode sequence to which the target polynucleotide is joined. A
barcode can, for example, comprise a nucleic acid sequence that
when joined to a target polynucleotide serves as an identifier of
the sample from which the target polynucleotide was derived.
[0115] Various adaptor designs can be envisioned which can be
suitable for generation of amplification-ready products of probe
target region sequence regions/strands of interest. In some
embodiments the at least first adaptor can be single or double
stranded. For example, when double stranded the two strands of the
adaptor can be self-complementary, non-complementary or partially
complementary. Recently, many improvements have been made in
adaptor design that has reduced the occurrence of adapter dimer.
These improvements can include the use of nucleotide analogs and
structured oligonucleotides, and have allowed for use of higher
concentrations of oligonucleotides in ligation reactions. The
higher concentrations of adapters in ligation reactions have
enabled researchers to produce high quality libraries from as few
as 150 copies of genome. Ligation of adaptors to the ends of DNA
fragments, in particular those fragments containing the regions of
interest can be suitable for carrying out the methods of the
invention. Various ligation modalities can be envisioned, dependent
on the choice of nucleic acid modifying enzymes and the resulting
double-stranded DNA cleavage. For example, when a blunt end product
comprising the target region/sequence of interest is generated,
blunt end ligation can be suitable. Alternatively, where the
cleavage can be carried out using a restriction enzyme of known
sequence specificity, leading to the generation of cleavage sites
with known sequence overhangs, suitable ends of the adaptors can be
designed to enable hybridization of the adaptor to the cleavage
site of the sequence region of interest and subsequent ligation.
Reagents and methods for efficient and rapid ligation of adaptors
are commercially available and are known in the art.
Nucleic Acid Modifying Enzymes
[0116] The nucleic acid (NA)-modifying enzyme can be DNA-specific
modifying enzyme. The NA-modifying enzyme can be selected for
specificity for double-stranded DNA. The enzyme can be a
duplex-specific endonuclease, a blunt-end frequent cutter
restriction enzyme, or other restriction enzyme. Examples of
blunt-end cutters can include Dral or Smal. The NA-modifying enzyme
can be an enzyme provided by NEW ENGLAND BIOLABS. The NA-modifying
enzyme can be a homing endonuclease (a homing endonuclease can be
an endonuclease that does not have a stringently-defined
recognition sequence). The NA-modifying enzyme can be a nicking
endonuclease (a nicking endonuclease can be an endonuclease that
can cleave only one strand of DNA in a double-stranded DNA
substrate). The NA-modifying enzyme can be a high fidelity
endonuclease (a high fidelity endonuclease can be an engineered
endonuclease that has less "star activity" than the wild-type
version of the endonuclease).
DNA-Dependent DNA Polymerases
[0117] DNA-dependent DNA polymerases for use in the methods and
compositions of the invention can be capable of effecting extension
of a probe target region or primer according to the methods of the
invention. In some embodiments, a DNA-dependent DNA polymerase can
be one that is capable of extending a probe target region, a
nucleic acid primer and the like in the presence of the DNA and/or
cDNA template. Exemplary DNA dependent DNA polymerases suitable for
the methods of the present invention include but are not limited to
Klenow polymerase, with or without 3'-exonuclease, Bst DNA
polymerase, Bsu polymerase, phi29 DNA polymerase, Vent polymerase,
Deep Vent polymerase, Taq polymerase, T4 polymerase, and E. coli
DNA polymerase 1, derivatives thereof, or to a mixture of
polymerases. In some cases, the polymerase does not comprise a
5'-exonuclease activity. In other cases, the polymerase comprises
5' exonuclease activity. In some cases, the primer or
oligonucleotide extension product of the present invention can be
performed using a polymerase comprising strong strand displacement
activity such as for example Bst polymerase. In other cases, the
primer extension of the present invention can be performed using a
polymerase comprising weak or no strand displacement activity. One
skilled in the art can recognize the advantages and disadvantages
of the use of strand displacement activity during the primer
extension step, and which polymerases can be expected to provide
strand displacement activity, see for example, Polymerases by NEW
ENGLAND BIOLABS.
Methods of Amplification
[0118] The methods, compositions and kits described herein can be
useful to generate amplification-ready products for downstream
applications such as massively parallel sequencing (i.e. next
generation sequencing methods), generation of libraries with
enriched population of sequence regions of interest, or
hybridization platforms. Methods of amplification are well known in
the art. Suitable amplification reactions can be exponential or
isothermal and can include any DNA amplification reaction,
including but not limited to polymerase chain reaction (PCR),
strand displacement amplification (SDA), linear amplification,
multiple displacement amplification (MDA), rolling circle
amplification (RCA), single primer isothermal amplification (SPIA,
see e.g. U.S. Pat. No. 6,251,639), Ribo-SPIA, or a combination
thereof. In some cases, the amplification methods for providing the
template nucleic acid can be performed under limiting conditions
such that only a few rounds of amplification (e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22,
23,24, 25,26, 27, 28, 29, 30 etc.), such as for example as can be
commonly done for cDNA generation. The number of rounds of
amplification can be about 1-30, 1-20, 1-15, 1-10, 5-30, 10-30,
15-30, 20-30, 10-30, 15-30, 20-30, or 25-30.
[0119] PCR is an in vitro amplification procedure based on repeated
cycles of denaturation, oligonucleotide primer annealing, and
primer extension by thermophilic template dependent polynucleotide
polymerase, resulting in the exponential increase in copies of the
desired sequence of the polynucleotide analyte flanked by the
primers. The two different PCR primers, which anneal to opposite
strands of the DNA, can be positioned so that the polymerase
catalyzed extension product of one primer can serve as a template
strand for the other, leading to the accumulation of a discrete
double stranded fragment whose length can be defined by the
distance between the 5' ends of the oligonucleotide primers.
Additional amplification methods are further described in U.S. Ser.
No. 13/750768 filed Jan. 25, 2013, incorporated by reference herein
in its entirety.
[0120] In some embodiments, the amplification can be exponential,
e.g. in the enzymatic amplification of specific double stranded
sequences of DNA by a polymerase chain reaction (PCR). In other
embodiments the amplification method can be linear. In other
embodiments the amplification method can be isothermal. Downstream
Applications
[0121] One aspect of the invention is that the methods and
compositions disclosed herein can be efficiently and
cost-effectively utilized for downstream analyses, such as next
generation sequencing or hybridization platforms, with minimal loss
of biological material of interest. The methods disclosed herein
can also be used in the analysis of genetic information of
selective genomic regions of interest (e.g., analysis of SNPs, copy
number variation, or other disease markers) as well as digital gene
expression from transcriptome analyses and genomic regions that can
interact with the selective region of interest. Sequencing
[0122] For example, the methods of the invention can be useful for
sequencing by the method commercialized by Illumina, as described
U.S. Pat. Nos. 5,750,341; 6,306,597; and 5,969,119. In general,
double stranded fragment polynucleotides can be prepared by the
methods of the present invention to produce amplified nucleic acid
sequences tagged at one (e.g., (A)/(A') or both ends (e.g.,
(A)/(A') and (C)/(C')). In some cases, single stranded nucleic acid
tagged at one or both ends can be amplified by the methods of the
present invention (e.g., by SPIA or linear PCR). The resulting
nucleic acid can then be denatured and the single-stranded
amplified polynucleotides can be randomly attached to the inside
surface of flow-cell channels. Unlabeled nucleotides can be added
to initiate solid-phase bridge amplification to produce dense
clusters of double-stranded DNA. To initiate the first base
sequencing cycle, four labeled reversible terminators, primers, and
DNA polymerase can be added. After laser excitation, fluorescence
from each cluster on the flow cell can be imaged. The identity of
the first base for each cluster can then be recorded. Cycles of
sequencing can be performed to determine the fragment sequence one
base at a time.
[0123] In some embodiments, the methods of the invention can be
useful for preparing target polynucleotides for sequencing by the
sequencing by ligation methods commercialized by Applied Biosystems
(e.g., SOLiD sequencing). In other embodiments, the methods can be
useful for preparing target polynucleotides for sequencing by
synthesis using the methods commercialized by 454/Roche Life
Sciences, including but not limited to the methods and apparatus
described in Margulies et al., Nature (2005) 437:376-380 (2005);
and U.S. Pat. Nos. 7,244,559; 7,335,762; 7,211,390; 7,244,567;
7,264,929; and 7,323,305. In other embodiments, the methods can be
useful for preparing target polynucleotide(s) for sequencing by the
methods commercialized by Helicos Biosciences Corporation
(Cambridge, Mass.) as described in U.S. application Ser. No.
11/167,046, and U.S. Pat. Nos. 7,501,245; 7,491,498; 7,276,720; and
in U.S. Patent Application Publication Nos. US20090061439;
US20080087826; US20060286566; US20060024711; US20060024678;
US20080213770; and US20080103058. In other embodiments, the methods
can be useful for preparing target polynucleotide(s) for sequencing
by the methods commercialized by Pacific Biosciences as described
in U.S. Pat. Nos. 7,462,452; 7,476,504; 7,405,281; 7,170,050;
7,462,468; 7,476,503; 7,315,019; 7,302,146; 7,313,308; and US
Application Publication Nos. US20090029385; US20090068655;
US20090024331; and US20080206764.
[0124] Another example of a sequencing technique that can be used
in the methods of the provided invention is semiconductor
sequencing provided by Ion Torrent (e.g., using the Ion Personal
Genome Machine (PGM)). Ion Torrent technology can use a
semiconductor chip with multiple layers, e.g., a layer with
micro-machined wells, an ion-sensitive layer, and an ion sensor
layer. Nucleic acids can be introduced into the wells, e.g., a
clonal population of single nucleic can be attached to a single
bead, and the bead can be introduced into a well. To initiate
sequencing of the nucleic acids on the beads, one type of
deoxyribonucleotide (e.g., dATP, dCTP, dGTP, or dTTP) can be
introduced into the wells. When DNA polymerase incorporates one or
more nucleotides, protons (hydrogen ions) can be released in the
well, which can be detected by the ion sensor. The semiconductor
chip can then be washed and the process can be repeated with a
different deoxyribonucleotide. A plurality of nucleic acids can be
sequenced in the wells of a semiconductor chip. The semiconductor
chip can comprise chemical-sensitive field effect transistor
(chemFET) arrays to sequence DNA (for example, as described in U.S.
Patent Application Publication No. 20090026082). Incorporation of
one or more triphosphates into a new nucleic acid strand at the 3'
end of the sequencing primer can be detected by a change in current
by a chemFET. An array can have multiple chemFET sensors.
[0125] Another example of a sequencing technique that can be used
in the methods of the provided invention is nanopore sequencing
(see e.g. Soni G V and Meller A. (2007) Clin Chem 53: 1996-2001). A
nanopore can be a small hole of the order of 1 nanometer in
diameter. Immersion of a nanopore in a conducting fluid and
application of a potential across it can result in a slight
electrical current due to conduction of ions through the nanopore.
The amount of current that flows is sensitive to the size of the
nanopore. As a DNA molecule passes through a nanopore, each
nucleotide on the DNA molecule obstructs the nanopore to a
different degree. Thus, the change in the current passing through
the nanopore as the DNA molecule passes through the nanopore can
represent a reading of the DNA sequence.
Genetic Analysis
[0126] The methods of the present invention can be used in the
analysis of genetic information of selective genomic regions of
interest as well as genomic regions that can interact with the
selective region of interest. Amplification methods as disclosed
herein can be used in the devices, kits, and methods known to the
art for genetic analysis, such as, but not limited to those found
in U.S. Pat. Nos. 6,449,562, 6,287,766, 7,361,468, 7,414,117,
6,225,109, and 6,110,709. In some cases, amplification methods of
the present invention can be used to amplify target nucleic acid of
interest for DNA hybridization studies to determine the presence or
absence of polymorphisms. The polymorphisms, or alleles, can be
associated with diseases or conditions such as genetic disease. In
other cases the polymorphisms can be associated with susceptibility
to diseases or conditions, for example, polymorphisms associated
with addiction, degenerative and age related conditions, cancer,
and the like. In other cases, the polymorphisms can be associated
with beneficial traits such as increased coronary health, or
resistance to diseases such as HIV or malaria, or resistance to
degenerative diseases such as osteoporosis, Alzheimer's or
dementia.
Digital Measurements
[0127] The methods of the present invention can be used in the
digital analysis of gene expression, gene expression patterns
associated with disease, including diagnosis, prognosis and
detection as well as identifying genetic disorders, e.g.,
chromosomal or gene translocations, deletions, duplications and
defects as well as studying selective genomic regions of interest
and genomic regions that can interact with the selective region of
interest. In some embodiments, determination of Digital Gene
Expression (DGE) or Copy Number Variation (CNV) digital
measurements can be achieved by quantitating the number of gene
reads within the total number of reads. In some embodiments, paired
end sequencing can be performed. Sequencing can be performed via
high throughput sequencing on a variety of platforms as is known to
one of skill in the art. In some embodiments, the sequencing
data/reads are mapped to the genome/transcriptome (for cDNA). In
some embodiments, sequence data can be evaluated to remove
duplicate reads. In some embodiments, probe sequences are counted
for the number of times they appear in de-duplicated sequence
dataset as a measure of the number of copies of the original
nucleic acid molecules present in the starting sample.
[0128] In some embodiments, verification of a probe correctly
annealing to its complementary probe target region within the
specific nucleic acid can be evaluated. In one embodiment,
evaluation of probe properly annealing can be done by paired end
alignment, if both ends, forward read and reverse read align as
expected, the probe is counted. In some embodiments, evaluation of
probe properly annealing can be done by examining the probe
sequence+20 bases sequenced of the specific nucleic acid 3' of the
probe sequence and use the forward read only for duplication
analysis. If probe+20 aligns, the probe was in the desired
location.
[0129] An advantage of using probe sequence counts rather than
random sequence is the simplification of copy number analysis
because the same sequences are used across different samples for
each measurement. Probe counting allows for high sample throughput
via multiplex sequencing (e.g., at least 96 samples per sequencing
run). Targeted RNA-sequencing can provide a high level of focus for
RNA-sequencing analysis, as greater than 90% of reads are derived
from targeted genes while extending the ability to target coding or
noncoding genes, specific exons, UTRs, RNA isoforms and gene
fusions. Probe counting can also reduce bias of exon usage,
transcript size and sequence dependent amplification/sequencing and
allow for removal of PCR duplicates.
[0130] The digital analysis can be performed by determining PCR
duplicates prior to quantitation. Such an analysis, using Illumina
sequencing technology, is illustrated in FIGS. 6A-6C and with
reference to FIG. 1. Briefly, the forward read, illustrated for
gDNA (FIG. 1), as shown in FIG. 6A includes a forward priming site
12 utilized by a forward primer 30 sequencing at least 1, at least
2, at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 15, at least 20, at
least 25, at least 30, at least 35 and so on base sequences of a
forward sequence 32 that extends into the specific nucleic acid 10
sequence and can be used to map forward read sequence 32 to the
genome (or transcriptome for cDNA) region. The index read, as shown
in FIG. 6B, can indicate the sample origin (e.g., a library barcode
common with the library). The index read starts at the index
priming site 18 with index primer 34 and includes the sequenced at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10 and so on bases
of the index bases (e.g., barcode sequence) 16 and n-random bases
14, yielding index read 36. In some embodiments, the forward read
sequence 32 in combination with indexing read base sequence and
N-random bases 36 are unique to the ligation event for each
specific nucleic acid sequence. In some embodiments, the
combination of the forward read sequence 32 start site genome
(transcriptome for cDNA) coordinates plus index read sequence 36
N-random bases 14 can be used to determine PGR duplicates for each
probe extension product 21 or 22 and thus the corresponding
specific nucleic acid sequence 10 or 9 having probe target region
50 or 60. The reverse read 44, as illustrated in FIG. 6C verities
the probe annealed to the correct genome/transcriptome position and
thus to its complementary probe target region. Flow cell sequences
33 and 35 are appended at the ends of the probe extension product
during enrichment.
[0131] The digital analysis can be performed by determining PGR
duplicates prior to quantitation. Such an analysis, is illustrated
in FIGS. 8A-8C and with reference to FIG. 1 and FIG. 5. The read
having the probe sequence 44, (FIG. 5) as illustrated in FIG. 8A
verifies the probe annealed to the correct genome/transcriptome
position and thus to its complementary probe target region. The
read having the probe sequence comprises a 15 base linker 38, a 40
base oligonucleotide gene specific sequence 50 or 60 (probe target
region) and an X-base (e.g., 10 base) of region 10 about 10 bases
3' to the 40 base oligonucleotide gene specific sequence 50 or 60
as represented in a genome (or transcriptome) database. The read
having the specific nucleic acid sequence 10, illustrated for gDNA
(FIG. 1), as shown in FIG. 8B includes a priming site 12 utilized
by primer 30 sequencing at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, at least 15, at least 20, at least 25, at least 30,
at least 35 and so on base sequences of sequence 32 (FIG. 5) that
extends into the specific nucleic acid 9 or 10 sequence and can be
used to map read having the specific nucleic acid sequence 32 to
the genome (or transcriptome for cDNA) region. The sequence read
comprising the index sequence and N6 sequence, as shown in FIG. 8C,
can indicate the sample origin (e.g., a library barcode common with
the library). The index read primer 34 anneals to the index priming
site 18 producing read sequence 36 comprising at least one of the
sequenced at least 1, at least 2, at least 3, at least 4, at least
5, at least 6, at least 7, at least 8, at least 9, at least 10 and
so on bases of the index bases (e.g., barcode sequence) 16 and
n-random bases 14, yielding sequencing read 36. In some
embodiments, the read having the specific nucleic acid sequence
(FIG. 8A) would verify specificity of the probe annealing to the
probe target region. In some embodiments, the read having the
specific nucleic acid sequence are binned into a probe target
sequence database and the e.g., 10 bases of specific nucleic acid
sequence 9 or 10 would need to align within a unique probe bin and
in so matching verify specificity of the probe annealing to its
probe target region. In some embodiments, the read having the
specific nucleic acid sequence would be compared with about 10 base
oligonucleotide matches determined if the sequence is unique within
the bin. Common reads would then be compared to the corresponding
N6 with identical N6 reads being collapsed together as a single
entry and only counted once. In some embodiments, the read having
at least one of the index read and the N6 read can be about 14
bases in length. In some embodiments, the read having the specific
nucleic acid sequence can be about 10 bases. In some embodiments,
the read having the probe sequence can be about 65 bases (about 15
bases for the linker sequence, about 40 bases for the probe target
region (gene specific sequence as represented in a
genome/transcriptome), and about 10 bases 3' to the probe target
region. In some embodiments, a look up table can be used. In some
embodiments, the probe sequences are counted. In some embodiments
the N6 sequence designates duplicates for elimination.
[0132] In some embodiments, read sequence 32 in combination with
read sequence 36 are unique to the ligation event for each specific
nucleic acid sequence. In some embodiments, the combination of read
sequence 32 start site genome (transcriptome for cDNA) coordinates
plus read sequence 36 N-random bases 14 can be used to determine
PGR duplicates for each probe extension product 22 or 21 and the
corresponding specific nucleic acid sequence 10 or 9 having probe
target region 50 or 60. In some embodiments, read sequence 44, as
illustrated in FIG. 6C verifies the probe annealed to the correct
genome/transcriptome position and also to its complementary probe
target region 50 or 60.
[0133] In some embodiments, the duplicate reads are removed prior
to DGE or CNV quantitation as disclosed above. The probe sequences
that are correctly mapped in the genome/transcriptome are then
counted. In some embodiments DGE or CNV can be determined by the
counts of each probe sequence. In some embodiments, probe counts
can be combined by, e.g., averaging counts across probes over the
length of a gene. In some embodiments, read counts can be
normalized between samples, e.g., read counts normalized as a
percentage of total reads. In some embodiments, read counts can be
normalized by e.g., normalizing total read counts before counting
each probe sequence. In some embodiments, read counts can be
normalized by the number of reads aligned to the genome or reads
derived from the probe target region,
[0134] As illustrated in FIG. 5 the structure of the sequencing
library and identification of sequencing reads provides for
multiplex quantitation using high throughput sequencing methods. As
illustrated in FIG. 7A. with reference to FIG. 6, forward primer 30
can be complementary to the forward read 1 priming site 12 and the
read can be extended 32 into the specific nucleic acid 10 (gDNA) or
13 (cDNA) sufficiently to map the read to the genome or
transcriptome. Additionally, an index read sequence illustrated in
FIG. 7B, can be read from the "index priming site" 18 using
complementary primer 34 and reading 36 into "index bases" 16 and
"n-random bases" 14, Additionally, a "reverse read sequence" can be
determined (FIG. 5, FIG. 6 and FIG. 7C). The reverse sequence
primer 42 hybridizes to the "reverse read priming site" 38 and
reads 44 through the probe 50 or 60 (gDNA or cDNA, respectively,
FIG. 1, FIG. 2, illustrated in FIG. 6) (probe target region site)
and at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10 and so on
adjacent bases 10 (gDNA) or 13 (cDNA) to verify if the probe
extension product 22 or 21 (gDNA or cDNA, respectively, FIG. 1,
FIG. 2) was the product of the probe hybridizing to the correct
genome or transcriptome. Probe sequences that are correctly mapped
in the genome or transcriptome are counted. DGE or CNV can be
determined by the counts of each probe sequence and/or probe counts
can be combined, including but not limited to, with averaging
counts across probes over the length of a gene corresponding to the
specific nucleic acid sequence. The read counts can be normalized
between samples. In some embodiments, read counts can be normalized
between samples, e.g., read counts normalized as a percentage of
total reads. In some embodiments, read counts can be normalized by
e.g., normalizing total read counts before counting each probe
sequence. In some embodiments, read counts can be normalized by the
number of reads aligned to the genome or reads derived from the
probe target region. Other methods for normalization are well known
to one of skill in the art of NGS sequence analysis.
[0135] FIG. 9 provides a graphical illustration of the construction
of sequencing libraries generated for digital analyses.
[0136] FIG. 10 provides a graphical illustration for the analyses
of sequencing data generated from sequencing libraries constructed
as illustrated in FIG. 9.
[0137] In some embodiments, the methods of the disclosed invention
can be used for digital measurements to analyze gene expression
characteristics and properties of, for example, but not limited to,
a tissue, a tumor, a circulating cell, as well as to compare
diseased verses non-diseased patients, and a patient's normal
verses diseased tissue. In some embodiments, the methods of the
disclosed invention can be used for copy number variation (CNV)
digital quantitation. CNV can indicate DNA alterations within a
genome resulting in a cell having an abnormal or a normal variation
in the number of copies of DNA sections. CNVs can identify deletion
of a large region of a genome resulting in fewer than normal number
or duplication of a large region of a genome having more than the
usual number within a chromosome. There are associations between
CNVs and susceptibility or resistance to disease. Such measurements
can be useful for diagnosis, disease staging, prognosis,
determining disease progression, viral load, as well as the impact
of gene expression or CNV on a therapeutic agent's efficacy or
efficiency and the like as would be known to one of skill in the
art.
[0138] In another aspect, disclosed is a composition comprising the
first nucleic acid fragment sequence amplified by the disclosed
method. In some embodiments, the first nucleic acid fragment or
non-fragmented nucleic acid sample can be from a human sample
selected from a same human: a single cell, a non-diseased tissue, a
diseased tissue, a FFPE sample or a fresh-frozen sample, a tissue,
an organ, a tumor, a specimen of an organic fluid taken from a
patient, freely circulation nucleic acid, a fungus, a prokaryotic
organism and a virus. In some embodiments, the second nucleic acid
fragment or non-fragmented nucleic acid sample can be from a sample
selected from the same human having tissue which can be either a
diseased tissue or a non-disease tissue, can be collected on a same
day, can be collected on separate days, can be collected from
different samples, can be collected from samples prepared by
different methods or can be collected from samples by different
purification methods and combinations thereof. In some embodiments,
the first nucleic acid fragment or non-fragmented nucleic acid
sample comprising the first adaptor sequence can be further
enriched and prepared for massively parallel sequencing. In some
embodiments, the first nucleic acid fragment or non-fragmented
nucleic acid sample can be double stranded. In some embodiments,
the first adaptor sequence can be appended to a 5' end of said
first nucleic acid fragment or non-fragmented nucleic acid sample.
In some embodiments, the first adaptor sequence comprises a
restriction and/or cleavage site for a nucleic acid modifying
enzyme.
[0139] In yet another aspect, a disclosed method can have a second
human nucleic acid fragment or second non-fragmented nucleic acid
sample with an adaptor. In some embodiments, the second human
sample can be derived from a different human than the human from
whom the first nucleic acid sample was derived. In some
embodiments, the second nucleic acid fragment or second
non-fragmented nucleic acid sample can be a sample selected from
the same human having tissue which can be either a diseased tissue
or a non-disease tissue, can be collected on a same day, can be
collected on separate days, can be collected from different
samples, can be collected from samples prepared by different
methods or can be collected from samples by different purification
methods and combinations thereof. In some embodiments, the second
nucleic acid fragment or second non-fragmented nucleic acid sample
can be a sample selected from a different human having tissue which
can be either a diseased tissue or a non-disease tissue, can be
collected on a same day, can be collected on separate days, can be
collected from different samples, can be collected from samples
prepared by different methods or can be collected from samples by
different purification methods and combinations thereof.
[0140] In a further aspect, disclosed is a method for quantitating
a second human nucleic acid according to previously disclosed the
methods.
Kits
[0141] Any of the compositions described herein can be included in
a kit. In a non-limiting example the kit, in suitable container
means, comprises: one adapter with a known sequence, one probe
having a sequence specific portion and common portion of known
sequence, one forward primer having a direct partial complement to
the at least either the adaptor or probe common portion and one
reverse primer having a direct partial complement to either the
adaptor or probe common portion. The kit can further contain
additional adapters, primers and/or reagents useful for ligation,
target enrichment and library preparation. The kit can further
optionally contain a DNA-polymerase. The kit can further optionally
contain reagents for amplification, for example reagents useful for
PCR amplification methods. The kit can further optionally contain
reagents for sequencing, for example, reagents useful for
next-generation massively parallel sequencing methods.
[0142] The containers of the kits can include at least one vial,
test tube, flask, bottle, syringe or other containers, into which a
component can be placed, and preferably, suitably aliquoted. Where
there is more than one component in the kit, the kit also can
contain a second, third or other additional container into which
the additional components can be separately placed. However,
various combinations of components can be included in a
container.
[0143] When the components of the kit can be provided in one or
more liquid solutions, the liquid solution can be an aqueous
solution. However, the components of the kit can be provided as
dried powder(s). When reagents and/or components are provided as a
dry powder, the powder can be reconstituted by the addition of a
suitable solvent.
[0144] A kit can include instructions for employing the kit
components as well the use of any other reagent not included in the
kit. Instructions can include variations that can be
implemented.
EXAMPLES
Example I
Differential Expression Levels of Specific Transcripts Between Two
Samples
[0145] Starting with 100 ng total RNA double stranded cDNA was made
using cDNA target enrichment module sold under the trademark
OVATION by NuGEN according to the manufacturer's recommendation.
cDNA samples were added directly into target enrichment kit sold
under the trademark OVATION by NuGEN according to manufacturer's
directions. Probes used in hybridization were a pool of probes
targeting 270 genes
[0146] The resulting libraries were diluted to 2 nM and paired end
sequencing was performed on the enriched library on an
Illumina.RTM. MiSeq.RTM. DNA Sequencer. The following paired end
series was run at 75 bases forward read (111), 75 bases reverse
read (R2), 14 base index reads (II).
Data Analysis
[0147] Paired end alignments were performed for the forward and
reverse sequencing reads and each were mapped to the human genome
version hgl9 using TopHat Alignment software (v.2.0.10) with
default settings. Pairs of reads that did not map to a targeted
region were eliminated. Forward reads with the same start
coordinate were then evaluated for the index sequence's N6 sequence
(n-random sequence). In instances where the N6 sequences were
identical, then the reads were marked as duplicates and only one
read of the group was retained as being derived from a single
distinct nucleic acid molecule. The identified duplicate reads were
marked and then removed. After filtering for on target and
deduplicated read pairs, the filtered reverse read sequences were
trimmed to remove adaptor and linker sequences using Trimgalore
(v.0.3.1), and shortened to the first 35 bases using FASTX Trimmer
software. Trimmed reverse read sequences were then mapped to a
probe sequence file (provided with the probes used) containing the
sequences of the targeting oligonucleotide using Bowtie Alignment
software (v. 1.0.0) with default parameters and `--norc` to prevent
reverse complement matching. Aligned reverse reads were associated
with their originating primer and counted. The number of times each
probe was detected was a measure of the number of times the
specific transcript was present in the original sample. Table 1
illustrates DGE data in which the read counts were normalized
between samples.
TABLE-US-00001 TABLE 1 Representation of gene expression of three
genes between two cancer cell lines Normalized Probe Normalized
Probe Gene Reads UHR Reads H2228 Ratio UHR/H2228 CCND3 499 494 1.01
TAF15 541 1074 0.52 PBX1 118 23 5.13
[0148] As depicted in Table 1, a mixed cancer cell line RNA sample
(UHR, Universal Human Reference RNA) has a relatively low level of
expression of genes TAF15 compared to H2228 cells (adenocarcinoma;
non-small cell lung cancer). Both cell types have very similar
expression levels of CCND3. Conversely UHR has higher expression of
PBX1 compared to H2228.
Example II
[0149] Differential Expression Levels of Specific Transcripts
Between Two Samples Without Genome Alignment Starting with IOOng
total RNA double stranded cDNA is made using cDNA target enrichment
module sold under the trademark OVATION by NuGEN, according to the
manufacturer's recommendation. cDNA samples are added directly into
the target enrichment kit sold under the trademark OVATION by NuGEN
according to manufacturer's directions. Probes used in
hybridization are a pool of probes targeting 270 genes, such as the
target enrichment module sold under the trademark OVATION by
NuGEN.
[0150] The resulting libraries are diluted to 2 nM and paired end
sequencing is performed on the enriched library on an Illumina.RTM.
MiSeq.RTM. DNA Sequencer. The following paired end series is run at
75 bases forward read (R1), 75 bases reverse read (R2), 14 base
index reads (II). Data Analysis
[0151] Reverse read sequences are trimmed with a pattern match at
the 5' end for 15 bp linker sequence and 0-3bases of diversity
sequence. After linker trimming the first 40 bp of the reverse read
are constructed into a Burrows-Wheeler transform (BWT) to match
tiled probe 12-mers in each read using BEETL software (version
1.1.0, github.com/BEETL/BEETL). Each read pair is then labeled as
being derived from the probe with the most 12-mer matches to the
reverse read. Labeled read pairs are then deduplicated per-probe,
by analyzing the index sequence's N6 sequence (n-random sequence)
along with the first 10 bases of the Forward read. In instances
where the reads are derived from the same probe, the N6 sequences
are identical, and the first 10 bases of the forward read are the
same, then the reads are marked as duplicates and only one read of
the group was retained as being derived from a single distinct
nucleic acid molecule. After filtering for deduplicated read pairs,
a total deduped read count was obtained for each probe. The number
of times each probe was detected was a measure of the number of
times the specific probe was present in the original sample. Counts
per probe are then averaged to obtain counts per gene based on the
probe annotation file, as a measure of relative abundance for that
gene in the particular sample.
Example III
Differential Expression of Specific Transcripts Mapping Forward
Reads
[0152] Starting with 100 ng total RNA input from Universal Human
Reference Sample (UHR) double stranded cDNA was made using the cDNA
target enrichment module sold under the trademark OVATION by NuGEN
according to the manufacturer's recommendation. cDNA samples were
added directly into the target enrichment kit sold under the
trademark OVATION by NuGEN according to manufacturer's directions.
A control library starting with 100 ng DNA input from Promega Male
Reference Sample was also processed using the target enrichment kit
sold under the trademark OVATION by NuGEN according to the
manufacturer's recommendation. Probes used in hybridization were a
pool of probes targeting 95 genes.
[0153] The resulting libraries were diluted to 2 nM and paired end
sequencing was performed on the enriched library on an
Illumina.RTM. MiSeq.RTM. DNA Sequencer. The following paired end
series was run at 70 bases forward read (R1), 88 bases reverse read
(R2), 14 base index reads (II).
Data Analysis
[0154] For both RNA and DNA derived data forward reads were quality
trimmed and trimmed of linker and adaptor sequences. For DNA
derived data, forward reads were mapped to the human genome version
hgl9 using Bowtie Alignment software with -m 2 parameter. For RNA
derived data, forward reads were first mapped to ribosomal RNA
reference using STAR Alignment software, reads that were unmapped
to ribosomal RNA were then mapped to the human version hgl9 also
using STAR Alignment software. After alignment, forward reads for
both the RNA and DNA data with the same start coordinate were then
evaluated for the index sequence's N6 sequence (n-random sequence).
In instances where the N6 sequences were identical, then the reads
were marked as duplicates and only one read of the group was
retained as being derived from a single distinct nucleic acid
molecule. The identified duplicate reads were marked and then
removed. CoverageBed software with default settings was then used
to count deduplicated forward reads overlapping any portion of each
target region (exons) for each dataset. The counts for each target
region were normalized for total reads in all target regions of the
dataset and then target regions corresponding to each exon within a
gene were averaged for a normalized gene count for the DNA and RNA
data. The DNA counts are expected to be quite even as expression
levels do not affect the probe's ability to generate reads. The RNA
counts are expected to have variability due to expression level
changes. Based on that idea, the log 2 ratio of normalized counts
RNA/DNA was then computed as a measure of gene abundance in the
RNA. A students T-test was then used to compute a p-value for each
gene measurement. Genes with a p-value<0.05 and a log ratio>0
were noted as upregulated genes and genes with a p-value<0.05
and a log ratio<0 were noted as downregulated genes.
[0155] Table 2 depicts five genes that were significantly
upregulated and five genes that were significantly downregulated
from the plot all gene abundance at the RNA level in the panel of
95 genes in chromosomal order (FIG. 11).
TABLE-US-00002 TABLE 2 Relative abundance of significantly changed
genes. Direction Gene Chromosome Abundance P-Value Up GUSB 7
6.291068 3.18E-04 Up ANXA1 9 8.132735 3.37E-07 Up ITGB7 12 13.091
5.98E-05 Up GAS6 13 5.678613 2.64E-04 Up TSC2 16 3.440623 3.22E-04
Down AMPD1 1 0.011096 2.95E-06 Down CR2 1 0.133867 3.35E-04 Down
ITGAX 16 0.194365 2.39E-06 Down NOS2 17 0.037126 9.74E-07 Down
ITGA2B 17 0.15729 2.31E-13
Example IV
Relative Expression Levels of Specific Transcripts Using Forward
Reads
[0156] Starting with 100 ng total RNA double stranded cDNA is made
using the cDNA target enrichment module sold under the trademark
OVATION by NuGEN according to the manufacturer's recommendation. An
adapter corresponding to the ILMN reverse flow cell sequence is
ligated onto the 5' end of each cDNA fragment. Probes containing a
sequence specific region followed by a 15 base linker and a XX base
sequence corresponding to the ILMN forward flow cell sequence are
annealed to the target and extended with a DNA polymerase. DNA
fragments containing both forward and reverse flow cell sequences
are amplified by PCR under conditions and with reagents recommended
and provided by NUGEN.
[0157] The resulting libraries are diluted to 2 nM and paired end
sequencing is performed on the enriched library on an Illumina.RTM.
MiSeq.RTM. DNA Sequencer. The following paired end series is run at
70 bases forward read (R1), and 14 base index reads (II).
Data Analysis
[0158] Forward reads sequences are trimmed to remove linker
sequences using Trimgalore software (v.0.3.1), and shortened to the
last 55 bases using FASTX Trimmer software. Trimmed forward reads
are mapped to the human genome version hgl9 using Bowtie Alignment
software with -m 2 parameter. Reads that do not map to a targeted
region are eliminated. Reads are identified that map to the same
start coordinates in the genome. Reads with the same start
coordinates are then evaluated for the index sequence's N6 sequence
(n-random sequence). In instances where the N6 sequences are
identical, then the read pairs are marked as duplicates and only
counted as being derived from a single distinct nucleic acid
molecule. The identified duplicate reads are marked and then
removed. Remaining reads are mapped to a probe sequence file
(provided with the probes used) containing the sequences of the
targeting oligonucleotide using Bowtie Alignment software (v.
1.0.0) with default parameters and norc' to prevent reverse
complement matching. The number of times each probe is detected is
a measure of the number of times the specific transcript is present
in the original sample. Reads overlapping any portion of the target
region (exons) are counted. The counts corresponding to each exon
within a gene are averaged. If any exon has counts below 2 standard
deviations of the average, that exon is dropped and the average
recalculated.
Example V
Determination of Copy Number Variation (CNV) by DNA Sequencing
[0159] Two human gDNA samples, one derived from a trisomy
chromosome 13 male and another a disomy chromosome 13 female were
fragmented to approximately 500 bp length by sonication with a
Covaris system. 100 ng of 500 bp fragments of gDNA from each sample
were added to the target enrichment kit sold under the trademark
OVATION by NuGEN according to manufacturer's directions. Probes
used in hybridization were a pool of probes targeting 344 genes,
such as the cancer panel target enrichment system sold under the
trademark OVATION by NuGEN.
[0160] The resulting libraries were diluted to 2nM and paired end
sequencing was performed on the enriched library on an
Illumina.RTM. MiSeq.RTM. DNA Sequencer. The following paired end
series was run at 75 bases forward read (R1), 88 bases reverse read
(R2), 14 base index read (II).
Data Analysis
[0161] Data were analyzed by two independent methods; removing
duplicates and not removing duplicates. Briefly, forward reads were
aligned to the human genome version hgl9 using Bowtie Alignment
software (v. 1.0.0) with default settings. If any forward reads
were determined to align to the same genomic start coordinate, the
corresponding index read was examined. In instances where the index
Read sequences corresponding to those forward reads with the same
genomic start coordinates were identical, the reads were marked as
duplicates and only counted as a single distinct nucleic acid
molecule. Reverse reads corresponding to the remaining distinct
forward reads were aligned using Bowtie to the sequences in a Probe
Database. Aligned reverse reads were binned and counted according
to which probe sequence they represent. The number of times each
probe was represented was a measure of the number of times the
starting specific nucleic acid molecule was present in the original
sample.
[0162] Alternatively, representation was established without
removing duplicate reads by cataloging the 40 base reverse reads
according to sequences present in the Probe Database. The number of
reads aligning to each representative in the probe reference
database was determined. Reads that did not match sequences in the
database were disregarded. The number of times each probe was
detected was a measure of the number of times the specific sequence
was present in the original sample. Table 3 depicts CNV data using
either method described above in which the read counts were
normalized to total sequencing read number and any counts below 10
were removed from analysis. The ratio of the probe count for a
given probe in a trisomy male sample to the counts of the same
probe in a wild type female sample were averaged for all probes on
a given chromosome.
TABLE-US-00003 TABLE 3 Copy number variation data from a trisomy 13
male in which read counts were normalized to total sequencing read
numbers No duplicate removal Duplicates removed Chromosome Average
probe count ratio Average probe count ratio chr 1 1.002485
0.990606365 chr 2 1.025382 1.010290049 chr 3 1.028736 1.016439439
chr 4 1.045166 1.032544903 chr 5 1.002378 0.998957554 chr 6
1.015266 0.997262904 chr 7 1.022412 1.021639631 chr 8 1.046251
1.028980962 chr 9 1.009415 0.991277289 chr 10 1.035216 0.993768193
chr 11 1.01177 1.00377304 chr 12 1.027063 1.004790487 chr 13
1.485411 1.471641235 chr 14 0.996186 0.986919321 chr 15 0.986867
0.981480187 chr 16 0.967682 0.964463441 chr 17 0.999821 0.992014077
chr 18 1.035764 1.016860381 chr 19 0.967125 0.958202381 chr 20
1.012836 1.010031227 chr 21 1.00104 1.013150115 chr 22 0.975676
0.972111601 chrX 0.548004 0.54329808
[0163] As depicted in Table 3, a diploid male has a single
X-chromosome vs. a diploid normal (wild type, WT) female having two
X-chromosomes which is identified by the 0.54 ratio (or 0.55 when
duplicates are not removed) of probe counts on the X chromosome
Likewise, both the male and female can have comparable normalized
counts for all other chromosomes with the exception of chromosome
13. The trisomy 13 male has an extra chromosome 13 as interpreted
by the 1.47 probe count ratio (or 1.49 when duplicates are not
removed) establishing a chromosome 13 copy number variation verses
comparison to the WT female.
Example VI
Determination of Copy Number Variation (CNV) in Cancer Cell Line by
DNA Sequencing
[0164] Two human gDNA samples, one derived from a pool of normal
male (Promega), and the other derived from that same pool of normal
male with two extra copies of EGFR and KIT genes spiked in for a
total of 4 copies each (previously validated by qPCR) were
fragmented to approximately 500 bp length by sonication with a
Covaris system. 100 ng of 500 bp fragments of gDNA from each sample
were added to the target enrichment kit sold under the trademark
OVATION by NuGEN according to manufacturer's directions. Probes
used in hybridization were a pool of probes targeting 509 genes,
such as the cancer panel target enrichment system sold under the
trademark OVATION by NuGEN.
[0165] The resulting libraries were diluted to 2nM and paired end
sequencing was performed on the enriched library on an
Illumina.RTM. MiSeq.RTM. DNA Sequencer. The following paired end
series was run at 70 bases forward read (R1), 88 bases reverse read
(R2), 14 base index read (II).
Data Analysis
[0166] For both datasets, forward reads in fastq format are trimmed
of linker sequence and low quality bases with Trim Galore software.
The reads were aligned to the human genome reference version hgl9
using Bowtie Alignment software (v 1.0.0) allowing for reads to map
to up to 2 places and picking only a single best alignment (-m
2--best). Aligned reads were subsequently deduplicated using the
deduplication software sold under the trademark NUDUP by NuGEN
(github.com/nugentechnologies/nudup). For deduplication, if any
reads are determined to align to the same genomic start coordinates
the corresponding index read is examined. In instances where the
index read sequences corresponding to those forward reads with the
same genomic start position were identical, the reads are marked as
duplicates and only a single read with the best quality from the
set is maintained.
[0167] The probes used in the enrichment experiment are expected to
produce reads that land within the starting coordinate of the probe
to approximately 300 bp downstream of the probe. For all probes in
the enrichment, the probe landing zone is defined in a bed file as
"probePlus300". The number of deduplicated reads within each
probePlus300 region are counted using BEDtools coverageBed. For
each probePlus300 region absolute counts are normalized by the
total deduplicated reads falling in all probePlus300 regions (sum
of all probePlus300 region counts) in order to compare counts
across experiments. Next, for each gene, or genomic region,
probePlus300 counts are averaged. Normalized average probePlus300
counts for each gene from the cell line sample are compared to the
normal blended male sample counts as a ratio. Furthermore, a
student's t-test can be used to compute genes or genomic regions
where the averaged probePlus300 counts are significantly different
for a given gene between the two samples with a multiple hypothesis
corrected p-value<0.005.
[0168] Table 4 depicts significant copy number changes and p-values
in the spike in sample. Specifically, there is only a significant
increase in copy number for EGFR and KIT genes--the two genes
spiked in at approximately 4 copies.
TABLE-US-00004 TABLE 4 Significant copy number changes and p-values
in the spike in sample. CNV GENE Chromosome Copies P-Value GAIN KIT
4 3.790214 5.05E-13 GAIN EGFR 7 4.059194 1.62E-16
Example VII
Rapid Library Generation for Determination of Copy Number Variation
by DNA Sequencing
[0169] Two human gDNA samples, one derived from a trisomy
chromosome 13 male and another a disomy chromosome 13 female can be
fragmented to approximately 500 bp length by sonication with a
Covaris system, lug of 500 bp fragments of gDNA from each sample
can be heat denatured at 95 C for 5 minutes in the presence of
probes and probe annealing solution, which may be provided in
target enrichment kits sold under the trademark OVATION by NuGEN,
and cooled at a rate of 0.1 C per minute to 60 C and held at that
temperature for at least 30 minutes. Following the annealing step,
a DNA polymerase and deoxynucleotides can be added to the solution
to extend probes annealed specifically to their template nucleic
acid. This solution can be cooled to room temperature and the
unincorporated probes removed by differential bead binding and
elution from SPRI beads, consistent with manufacturer's
recommendations. The recovered double stranded DNA can undergo end
repair and ligation with solutions provided in the target
enrichment kit sold under the trademark OVATION by NuGEN.
[0170] The resulting libraries can be diluted to 2 nM and paired
end sequencing performed on the enriched library on an
Illumina.RTM. MiSeq.RTM. DNA Sequencer. The following paired end
series can be run; 75 bases forward read (read 1), 75 bases reverse
read (read 2), 14 base index read (read 3).
Data Analysis
[0171] Data can be analyzed by cataloging the 75 base reverse reads
by sequences present in the Probe Database. The number of reads
aligning to each representative in the probe reference database can
be determined. Reads that did not align to sequences in the
database could be disregarded. The number of times each probe is
detected can be a measure of the number of times the specific
sequence was present in the original sample. Read counts can be
normalized to total sequencing read number and any counts below 10
can be removed from analysis. The ratio of the probe count for a
given probe in a trisomy male sample to the counts of the same
probe in a wild type female sample can be averaged for all probes
on a given chromosome.
[0172] Data from this test would reveal the male sample as having a
single X-chromosome vs. a diploid normal (wildtype, WT) female
having two X-chromosomes and therefore an approximate 0.5 ratio of
probe counts on the X chromosome. Likewise, both the male and
female would have comparable normalized counts for all other
chromosomes with the exception of chromosome 13. The trisomy 13
male has an extra chromosome 13, this would result in a probe count
ratio of approximately 1.5 probe count ratio relative to the WT
female.
[0173] Those having ordinary skill in the art will understand that
many modifications, alternatives, and equivalents are possible. All
such modifications, alternatives, and equivalents are intended to
be encompassed herein.
[0174] While the principles of this invention have been described
in connection with specific embodiments, it can be understood
clearly that these descriptions are made only by way of example and
are not intended to limit the scope of the invention. What has been
disclosed herein has been provided for the purposes of illustration
and description. It is not intended to be exhaustive or to limit
what is disclosed to the precise forms described. Many
modifications and variations will be apparent to the practitioner
skilled in the art. What is disclosed was chosen and described in
order to best explain the principles and practical application of
the disclosed embodiments of the art described, thereby enabling
others skilled in the art to understand the various embodiments and
various modifications that are suited to the particular use
contemplated. It is intended that the scope of what is disclosed be
defined by the following claims and their equivalence.
* * * * *