U.S. patent application number 13/835093 was filed with the patent office on 2016-12-08 for method of sequence determination using sequence tags.
The applicant listed for this patent is Adaptive Biotechnologies Corp.. Invention is credited to Malek Faham, Martin Moorhead, Thomas Willis, Jianbiao Zheng.
Application Number | 20160355893 13/835093 |
Document ID | / |
Family ID | 49114450 |
Filed Date | 2016-12-08 |
United States Patent
Application |
20160355893 |
Kind Code |
A9 |
Faham; Malek ; et
al. |
December 8, 2016 |
METHOD OF SEQUENCE DETERMINATION USING SEQUENCE TAGS
Abstract
The invention is directed to the use of sequence tags to improve
sequence determination of amplicons of related sequences,
particularly large and complex amplicons, such as those comprising
recombined nucleic acids encoding immune receptor molecules. In one
aspect, sequence reads having the same sequence tags are aligned
after which final base calls are determined from a (possibly
weighted) average base call from sequence read base calls at each
position. Similarly, in another aspect, sequence reads comprising
series of incorporation signals are aligned by common sequence tags
and base calls in homopolymer regions are made as a function
incorporation signal values at each "flow" position.
Inventors: |
Faham; Malek; (Pacifica,
CA) ; Moorhead; Martin; (San Francisco, CA) ;
Willis; Thomas; (San Francisco, CA) ; Zheng;
Jianbiao; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Adaptive Biotechnologies Corp. |
Seattle |
WA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130236895 A1 |
September 12, 2013 |
|
|
Family ID: |
49114450 |
Appl. No.: |
13/835093 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13100365 |
May 4, 2011 |
8748103 |
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13835093 |
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12615263 |
Nov 9, 2009 |
8236503 |
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13100365 |
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61658317 |
Jun 11, 2012 |
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61738277 |
Dec 17, 2012 |
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61776647 |
Mar 11, 2013 |
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61332175 |
May 6, 2010 |
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61455743 |
Oct 25, 2010 |
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61446822 |
Feb 25, 2011 |
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61112693 |
Nov 7, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 1/6869 20130101; C12Q 1/6869 20130101; C12Q 1/6888 20130101;
C12Q 2525/191 20130101; C12Q 2563/179 20130101; C12Q 2537/143
20130101; C12Q 2600/158 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of determining clonotypes of an immune repertoire, the
method comprising the steps: (a) obtaining a sample from an
individual comprising T-cells and/or B-cells; (b) attaching
sequence tags to molecules of recombined nucleid acids of T-cell
receptor genes or immunoglobulin genes of the T-cells and/or
B-cells to form tag-molecule conjugates, wherein substantially
every molecule of the tag-molecule conjugates has a unique sequence
tag; (c) amplifying the tag-molecule conjugates; (d) sequencing the
tag-molecule conjugates; and (e) aligning sequence reads of like
sequence tags to determine sequence reads corresponding to the same
clonotypes of the repertoire.
2. The method of claim 1 wherein said step of aligning further
includes determining a nucleotide sequence of each of said
clonotype of each of said tag-molecule conjugate by determining a
majority nucleotide at each nucleotide position of said clonotypes
of said like sequence tags.
3. The method of claim 1 wherein said step of attaching includes
labeling by sampling said molecules of recombined nucleic
acids.
4. The method of claim 3 wherein said step of attaching is
implemented in a reaction mixture such that said sequence tags are
present in the reaction mixture in a concentration at least 100
time that of said molecules of recombined nucleic acid.
5. The method of claim 4 wherein said sequence tags are incorpored
into primers specific for said molecules of recombined nucleic
acids.
6. The method of claim 5 wherein said sequence tags are mosaic
tags.
7. A method of determining a number of lymphocytes in a sample, the
method comprising the steps of: (a) obtaining a sample from an
individual comprising lymphocytes; (b) attaching sequence tags to
molecules of recombined nucleic acids of T-cell receptor genes or
of immunoglobulin genes of the lymphocytes to form tag-molecule
conjugates, wherein substantially every molecule of the
tag-molecule conjugates has a unique sequence tag; (c) amplifying
the tag-molecule conjugates; (d) sequencing the tag-molecule
conjugates; (e) counting the number of distinct sequence tags to
determine the number of lymphocytes in the sample.
8. The method of claim 7 wherein said recombined nucleic acids are
DNA.
9. The method of claim 7 wherein said lymphocyte is a T-cell and
said recombined nucleic acids are T-cell receptor genes or
fragments thereof.
10. The method of claim 7 wherein said lymphocyte is a B-cell and
said recombined nucleic acids are immunoglobulin genes or fragments
thereof.
11. A method of determining clonotypes of an immune repertoire, the
method comprising the steps: (a) obtaining a sample from an
individual comprising T-cells and/or B-cells; (b) labeling by
sampling molecules from the T-cells and/or B-cells to form
tag-molecule conjugates, wherein each tag has a sequence and each
molecule comprises a recombined nucleic acid from a T-cell receptor
gene or an immunoglobulin gene; (c) sequencing the tag-molecule
conjugates; and (d) aligning sequence reads of like tags to
determine like clonotypes.
12. The method of claim 11 wherein said step of aligning further
includes determining a nucleotide sequence of each of said
clonotype of each of said tag-molecule conjugate by determining a
majority nucleotide at each nucleotide position of said clonotypes
of said like sequence tags.
13. The method of claim 11 wherein said step of attaching is
implemented in a reaction mixture such that said sequence tags are
present in the reaction mixture in a concentration at least 100
time that of said molecules of recombined nucleic acid.
14. A method of detecting clonotype carry over contamination in a
patient being monitored for minimal residual disease, the method
comprising the steps of: monitoring a patient for minimal residual
disease by periodically measuring a clonotype profile of the
patient in accordance with the method of claim 1; and recording
said sequence of each of said sequence tags of each measurement of
clonotype profiles; and detecting clonotype carry over
contamination if a sequence tag of any prior clonotype profile is
detected in a subsequent clonotype profile.
15. A method of determining nucleotide sequences of one or more
polynucleotides in one or more sequencing-by-synthesis reactions,
the method comprising the steps: (a) attaching a sequence tag to
each of the one or more polynucleotides to form tag-polynucleotide
conjugates, wherein substantially every polynucleotide of the
tag-polynucleotide conjugates has a unique sequence tag; (b)
amplifying the tag-polynucleotide conjugates; (c) sequencing by
synthesis amplified tag-polynucleotide conjugates, wherein
sequencing by synthesis comprises at least one dNTP flow; and (d)
determining for each tag-polynucleotide having the same sequence
tag a number of nucleotide incorporations for each dNTP flow as a
function of measured incorporation signals for each such dNTP
flow.
16. The method of claim 15 wherein said one or more polynucleotides
are a plurality of polynucleotides.
17. The method of claim 15 wherein said plurality is at least
10.sup.4.
18. The method of claim 15 wherein said number of nucleotide
incorporations for each of said dNTP flows is a whole number
closest to an average of said measured incorporation signals.
19. The method of claim 15 wherein said step of sequencing by
synthesis comprises the steps of: (a) forming for each of said
tag-polynucleotide conjugates a complex comprising a sequencing
primer, a nucleic acid polymerase, and a tag-polynucleotide
conjugate under conditions that permit annealing of the sequencing
primer to such tag-polynucleotide conjugate and extension of such
sequencing primer along the tag-polynucleotide conjugate in the
presence of nucleoside triphosphates by the nucleic acid
polymerase; (b) introducing a dNTP to the complex by a dNTP flow;
(c) measuring incorporation signals; (d) washing the complex; and
(e) repeating steps (b) through (d).
20. The method of claim 19 wherein said dNTPs are extension-blocked
dNTPs and wherein said step of washing further includes a step of
de-blocking incorporated extension-blocked dNTPs so that in a
subsequent step of introducing a further extension-blocked dNTP may
be incorporated.
21. The method of claim 15 wherein each different
tag-polynucleotide conjugate is in a different reaction confinement
region.
22. The method of claim 15 wherein said step of attaching includes
labeling by sampling said one or more polynucleotides to form said
tag-polynucleotide conjugates.
23. The method of claim 15 wherein said sequence tags are mosaic
tags having variable regions with lengths in the range of from 1 to
5 nucleotides.
24. The method of claim 15 wherein said sequence tags comprise
alternating regions comprising nucleotides selected from disjoint
subsets of A, C, G and T, such that each alternating region has a
length in the range of from 1 to 5 nucleotides.
25. The method of claim 15 wherein said step of sequencing
comprises sequencing a sample of said amplified tag-polynucleotide
conjugates.
26. The method of claim 15 wherein said one or more polynucleotides
are molecules of recombined nucleid acids of T-cell receptor genes
or immunoglobulin genes.
Description
[0001] This application is a continuation-in-part of co-pending
U.S. patent application Ser. No. 13/100,365 filed 4 May 2011, and
claims priority from the following U.S. provisional patent
applications: Ser. No. 61/658,317 filed 11 Jun. 2012; Ser. No.
61/738,277 filed 17 Dec. 2012; and Ser. No. 61/776,647 filed 11
Mar. 2013, which applications are each hereby incorporated by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Analysis of biological or medical samples often requires the
determination of nucleic acid sequences of large and complex
populations of DNA and/or RNA, e.g. Gloor et al, PLoS ONE 5(10);
e15406, (2010); Petrosino et al, Clinical Chemistry, 55(5):
8(56-866 (2009); Arstila et al, Science, 286: 958-961 (1999). In
particular, profiles of nucleic acids encoding immune molecules,
such as T cell or B cell receptors, or their components, contain a
wealth of information on the state of health or disease of an
organism, so that the use of such profiles as diagnostic or
prognostic indicators has been proposed for a wide variety of
conditions, e.g. Faham and Willis, U.S. patent publication
2010/0151471; Freeman et al, Genome Research, 19: 1817-1824 (2009);
Boyd et al, Sci. Transl. Med., 1(12): 12ra23 (2009); He et al,
Oncotarget (Mar. 8, 2011). Such sequence-based profiles are capable
of much greater sensitivity than approaches based on size
distributions of amplified target nucleic acids, sequence sampling
by microarrays, hybridization kinetics curves from PCR amplicons,
or other approaches, e.g. Morley et al, U.S. Pat. No. 5,418,134;
van Dongen et al. Leukemia, 17: 2257-2317 (2003); Ogle et al.
Nucleic Acids Research, 31: e139 (2003); Wang et al, BMC Genomics,
8: 329 (2007); Baum et al. Nature Methods, 3(11): 895-901 (2006).
However, the efficient determination of clonotypes and clonotype
profiles from sequence data poses challenges because of the size of
populations to be analyzed, the similarity of sequences in such
populations, the limited predictability of natural variability
among the sequences, and noise introduced into the data by a host
of sample preparation and measurement steps, e.g. Warren et al,
Genome Research, 21(5): 790-797 (2011).
[0003] Sequence tags, or barcodes, have been used in a variety of
ways to assist in the analysis of nucleic acid populations,
including labeling, contamination monitoring, rare mutant
detection, physical sorting, molecular counting, and the like, e.g.
Kinde et al. Proc. Natl. Acad. Sci., 108(23): 9530-9535 (2011);
Casbon et al. U.S. patent publication 2012/0071331; Brenner, U.S.
Pat. No. 5,635,440; Brenner and Macevicz, U.S. Pat. No. 7,537,897;
Brenner et al, Proc. Natl. Acad. Sci. 97: 1665-1670 (2000); Church
et al, European patent publication 0 303 459; Shoemaker et al.
Nature Genetics, 14: 450-456 (1996); Morris et al. European patent
publication 0799897A1; Wallace, U.S. Pat. No. 5,981,179. Recently
Kinde et al (cited above) showed how sequence tags could be used to
distinguish sequencing and amplification errors from rare mutations
in a reference sequence.
[0004] In view of the importance of accurate sequencing for medical
and diagnostic applications, it would be highly advantageous if the
use of sequence tags could be expanded for increasing the
efficiency and accuracy of sequence determination in such
applications.
SUMMARY OF THE INVENTION
[0005] The present invention is drawn to methods for producing
sequence-based profiles of complex nucleic acid populations,
particularly recombined nucleic acid populations encoding
repertoires of immune molecules or portions thereof. The invention
is exemplified in a number of implementations and applications,
some of which are summarized below and throughout the
specification.
[0006] In one aspect, the invention is directed to a method of
determining clonotypes of an immune repertoire comprising the
steps: (a) obtaining a sample from an individual comprising T-cells
and/or B-cells; (b) attaching sequence tags to molecules of
recombined nucleid acids of T-cell receptor genes or immunoglobulin
genes of the T-cells and/or B-cells to form tag-molecule
conjugates, wherein substantially every molecule of the
tag-molecule conjugates has a unique sequence tag; (c) amplifying
the tag-molecule conjugates: (d) sequencing the tag-molecule
conjugates; and (e) aligning sequence reads of like sequence tags
to determine sequence reads corresponding to the same clonotypes of
the repertoire.
[0007] In another aspect, the invention is directed to a method of
detecting clonotype carry over contamination in a patient being
monitored for minimal residual disease comprising the steps of: (a)
monitoring a patient for minimal residual disease by periodically
measuring a clonotype profile of the patient in accordance with the
method of claim AI; (b) recording said sequence tags of each
measurement of clonotype profiles; and (c) detecting clonotype
carry over contamination if a sequence tag of any prior clonotype
profile is detected in a subsequent clonotype profile.
[0008] In another aspect, the invention is directed to a method of
determining a number of lymphocytes in a sample comprising the
following steps: (a) obtaining a sample from an individual
comprising lymphocytes; (b) attaching sequence tags to molecules of
recombined nucleic acids of T-cell receptor genes or of
immunoglobulin genes of the lymphocytes to form tag-molecule
conjugates, wherein substantially every molecule of the
tag-molecule conjugates has a unique sequence tag; (c) amplifying
the tag-molecule conjugates: (d) sequencing the tag-molecule
conjugates; (e) counting the number of distinct sequence tags to
determine the number of lymphocytes in the sample.
[0009] In another aspect, the invention is directed to a method of
determining nucleotide sequences of one or more polynucleotides in
one or more sequencing-by-synthesis reactions, which method
comprises the steps; (a) attaching a sequence tag to each of the
one or more polynucleotides to form tag-polynucleotide conjugates,
wherein substantially every polynucleotide of the
tag-polynucleotide conjugates has a unique sequence tag; (b)
amplifying the tag-polynucleotide conjugates; (c) sequencing by
synthesis amplified tag-polynucleotide conjugates, wherein
sequencing by synthesis comprises at least one dNTP flow; and (d)
determining for each tag-polynucleotide having the same sequence
tag a number of nucleotide incorporations for each dNTP flow as a
function of measured incorporation signals for each such dNTP
flow.
[0010] The present invention provides methods for determining
clonotypes and clonotype profiles from large sets of sequence data
obtained by high throughput sequencing of somatically recombined
nucleic acids that encode immune molecules. In one aspect, the
invention implements the above methods by labeling each somatically
recombined nucleic acid molecule in a sample with a unique sequence
tag, which is used to group sequence reads containing copies of the
same clonotype sequence from the sample.
[0011] These above-characterized aspects, as well as other aspects,
of the present invention are exemplified in a number of illustrated
implementations and applications, some of which are shown in the
figures and characterized in the claims section that follows.
However, the above summary is not intended to describe each
illustrated embodiment or every implementation of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention is obtained by
reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0013] FIGS. 1A-1B illustrate an example of labeling by sampling to
attach unique sequence tags to nucleic acid molecules.
[0014] FIG. 1C illustrates an IgH transcript and sources of natural
variability within it.
[0015] FIGS. 2A-2C show a two-staged PCR scheme for amplifying
recombined nucleic acid molecules.
[0016] FIG. 3 illustrates one implementation of the step of
determining the sequence of a clonotype from sequence reads
associated with the same sequence tag.
[0017] FIGS. 4A-4D illustrate an embodiment of the invention
directed to determining homopolymer regions in
sequencing-by-synthesis operations.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques and descriptions of
molecular biology (including recombinant techniques),
bioinformatics, cell biology, and biochemistry, which are within
the skill of the art. Such conventional techniques include, but are
not limited to, sampling and analysis of blood cells, nucleic acid
sequencing and analysis, and the like. Specific illustrations of
suitable techniques can be had by reference to the example herein
below. However, other equivalent conventional procedures can, of
course, also be used. Such conventional techniques and descriptions
can be found in standard laboratory manuals such as Genome
Analysis: A Laboratory Manual Series (Vols. I-IV): PCR Primer: A
Laboratory Manual; and Molecular Cloning: A Laboratory Manual (all
from Cold Spring Harbor Laboratory Press); and the like.
[0019] In one aspect, the invention is directed to a method for
obtaining and analyzing sequence data from a repertoire of immune
molecules, such as T cell receptors (TCRs) or B cell receptors
(BCRs) or defined fragments thereof, to rapidly and efficiently
determine a clonotype profile. Sequence data typically comprises a
large collection of sequence reads, i.e. sequences of base calls
and associated quality scores, from a DNA sequencer used to analyze
the immune molecules. A key challenge in constructing clonotype
profiles is to rapidly and accurately distinguish sequence reads
that contain genuine differences from those that contain errors
from non-biological sources, such as the extraction steps,
sequencing chemistry, amplification chemistry, or the like. An
aspect of the invention includes attaching a unique sequence tag to
each clonotype in a sample to assist in determining whether
sequence reads of such conjugates are derived from the same
original clonotype. In accordance with one aspect of the invention,
sequence tags are attached to the somatically recombined nucleic
acid molecules to form tag-molecule conjugates wherein each
recombined nucleic acid of such a conjugate has a unique sequence
tag. Usually such attachment is made after nucleic acid molecules
are extracted from a sample containing T cells and/or B cells.
Preferably, such unique sequence tags differ as greatly as possible
from one another as determined by conventional distance measures
for sequences, such as, a Hamming distance, or the like. By
maximizing the distance between sequence tags in tag-molecule
conjugates, even with a high rate of sequencing and amplification
errors, a sequence tag of a conjugate remains far closer to its
ancestoral tag sequence than to that of any other tag sequence of a
different conjugate. For example, if 16-mer sequence tags are
employed and each such tag on a set of clonotypes has a Hamming
distance of at least fifty percent, or eight nucleotides, from
every other sequence tag on the clonotypes, then at least eight
sequencing or amplification errors would be necessary to transform
one such tag into another for a mis-read of a sequence tag (and the
incorrect grouping of a sequence read of a clonotype with the wrong
sequence tag). In one embodiment, sequence tags are selected so
that after attachment to recombined nucleic acids molecules to form
tag-molecule conjugates, the Hamming distance between tags of the
tag-molecule conjugates is a number at least twenty-five percent of
the total length of such sequence tags (that is, each sequence tag
differs in sequence from every other such tag in at least 25
percent of its nucleotides); in another embodiment, the Hamming
distance between such sequence tags is a number at least 50 percent
of the total length of such sequence tags.
[0020] In one aspect, the invention is implemented by the following
steps: (a) obtaining a sample from an individual comprising T-cells
and/or B-cells; (b) attaching sequence tags to molecules of
recombined nucleid acids of T-cell receptor genes or immunoglobulin
genes of the T-cells and/or B-cells to form tag-molecule
conjugates, wherein substantially every molecule of the
tag-molecule conjugates has a unique sequence tag; (c) amplifying
the tag-molecule conjugates; (d) sequencing the tag-molecule
conjugates; and (c) aligning sequence reads of like sequence tags
to determine sequence reads corresponding to the same clonotypes of
the repertoire. Samples containing B-cells or T-cells are obtained
using conventional techniques, as described more idly below. In the
step of attaching sequence tags, preferably sequence tags are not
only unique but also are sufficiently different from one another
that the likelihood of even a large number of sequencing or
amplification errors transforming one sequence tag into another
would be close to zero. After attaching sequence tags,
amplification of the tag-molecule conjugate is necessary for most
sequencing technologies; however, whenever single-molecule
sequencing technologies are employed an amplification step is
optional. Single molecule sequencing technologies include, but are
not limited to, single molecule real-time (SMRT) sequencing,
nanopore sequencing, or the like, e.g. U.S. Pat. Nos. 7,313,308;
8,153,375; 7,907,800; 7,960,116; 8,137,569; Manrao et al, Nature
Biotechnology, 4(8): 2685-2693 (2012): and the like.
[0021] In another aspect, the invention includes a method for
determining the number of lymphocytes in a sample by counting
unique sequence tags. Even without sequence tags, clonotypes of
TCR.beta. or IgH genes, particularly those including the V(D)J
regions, provide for a lymphocyte and its clones a unique marker.
Whenever recombined nucleic acids are obtained from genomic DNA,
then a count of lymphocytes in a sample may be estimated by the
number of unique clonotypes that are counted after sequencing. This
approach breaks down whenever there are significant clonal
populations of identical lymphocytes associated with the same
clonotype (or when recombined nucleic acids are obtained from mRNA
of a sample, whose quantity of individual sequences may reflect, or
depend on, expression rate as well as cell number). The use of
sequence tags overcomes this short coming and is especially useful
for providing counts of lymphocytes in patients suffering from many
lymphoid disorders, such as lymphomas or leukemias. In accordance
with one aspect of the invention, sequence tags may be used to
obtain an absolute count of lymphocytes in a sample regardless of
whether there is a large dominant clone present, such as with
leukemia. Such a method may be implemented with the steps: (a)
obtaining a sample from an individual comprising lymphocytes: (b)
attaching sequence tags to molecules of recombined nucleic acids of
T-cell receptor genes or of immunoglobulin genes of the lymphocytes
to form tag-molecule conjugates, wherein substantially every
molecule of the tag-molecule conjugates has a unique sequence tag;
(c) amplifying the tag-molecule conjugates; (d) sequencing the
tag-molecule conjugates; and (e) counting the number of distinct
sequence tags to determine the number of lymphocytes in the sample.
In some embodiments, the molecules of recombined nucleic acids are
from genomic DNA.
[0022] In one embodiment of the invention, sequence tags are
attached to recombined nucleic acid molecules of a sample by
labeling by sampling, e.g. as disclosed by Brenner et al, U.S. Pat.
No. 5,846,719; Brenner et al, U.S. Pat. No. 7,537,897; Macevicz,
International patent publication WO 2005/111242; and the like,
which are incorporated herein by reference. In labeling by
sampling, polynucleotides of a population to be labeled (or
uniquely tagged) are used to sample (by attachment, linking, or the
like) sequence tags of a much larger population. That is, if the
population of polynucleotides has K members (including replicates
of the same polynucleotide) and the population of sequence tags has
N members, then N>>K. In one embodiment, the size of a
population of sequence tags used with the invention is at least 10
times the size of the population of clonotypes in a sample; in
another embodiment, the size of a population of sequence tags used
with the invention is at least 100 times the size of the population
of clonotypes in a sample; and in another embodiment, the size of a
population of sequence tags used with the invention is at least
1000 times the size of the population of clonotypes in a sample. In
other embodiments, a size of sequence tag population is selected so
that substantially every clonotype in a sample will have a unique
sequence tag whenever such clonotypes are combined with such
sequence tag population, e.g. in an attachment reaction, such as a
ligation reaction, amplification reaction, or the like. In some
embodiments, substantially every clonotype means at least 90
percent of such clonotypes will have a unique sequence tag: in
other embodiments, substantially every clonotype means at least 99
percent of such clonotypes will have a unique sequence tag; in
other embodiments, substantially every clonotype means at least
99.9 percent of such clonotypes will have a unique sequence tag. In
many tissue samples or biopsies the number of T cells or B cells
may be up to or about 1 million cells: thus, in some embodiments of
the invention employing such samples, the number of unique sequence
tags employed in labeling by sampling is at least 10.sup.8 or in
other embodiments at least 10.sup.9.
[0023] In such embodiments, in which up to 1 million clonotypes are
labeled by sampling, large sets of sequence tags may be efficiently
produced by combinatorial synthesis by reacting a mixture of all
four nucleotide precursors at each addition step of a synthesis
reaction, e.g. as disclosed in Church. U.S. Pat. No. 5,149,625,
which is incorporated by reference. The result is a set of sequence
tags having a structure of "N.sub.1N.sub.2 . . . N.sub.k" where
each N=A, C, G or T and k is the number of nucleotides in the tags.
The number of sequence tags in a set of sequence tags made by such
combinatorial synthesis is 4.sup.k. Thus, a set of such sequence
tags with k at least 14, or k in the range of about 14 to 18, is
appropriate for attaching sequence tags to a 10.sup.6-member
population of molecules by labeling by sampling. Sets of sequence
tags with the above structure include many sequences that may
introduce difficulties or errors while implementing the methods of
the invention. For example, the above combinatorially synthesized
set of sequence tags includes many member tags with homopolymers
segments that some sequencing approaches, such as
sequencing-by-synthesis approaches, have difficulty determining
with accuracy above a certain length. Therefore, the invention
includes combinatorially synthesized sequence tags having
structures that are efficient for particular method steps, such as
sequencing. For example, several sequence tag structures efficient
for sequencing-by-synthesis chemistries may be made by dividing the
four natural nucleotides into disjoint subsets which are used
alternatively in combinatorial synthesis, thereby preventing
homopolymer segments above a given length. For example, let z be
either A or C and x be either G or T, to give a sequence tag
structure of [0024] [(z).sub.1(z).sub.2 . . .
(z).sub.i][(x).sub.1(x).sub.2 . . . (x).sub.j] . . . where i and j,
which may be the same or different, are selected to limit the size
of any homopolymer segment. In one embodiment, i and j are in the
range of from 1 to 6. In such embodiments, sequence tags may have
lengths in the range of from 12 to 36 nucleotides; and in other
embodiments, such sequence tags may have lengths in the range of
from 12 to 24 nucleotides. In other embodiments other pairing of
nucleotides may be used, for example, z is A or T and x is G or C;
or z is A or G and x is T or C. Alternatively, let z' be any
combination of three of the four natural nucleotides and let x' be
whatever nucleotide is not a z' (for example, z' is A, C or G, and
x' is T). This gives a sequence tag structure as follows: [0025]
[(z').sub.1(z').sub.2 . . . (z').sub.i]x'[(z').sub.1(z').sub.2 . .
. (z').sub.i]x' . . . where i is selected as above and the
occurrence of x' serves as a punctuation to terminate any undesired
homopolymers.
Further Sequence Tags
[0026] The invention uses methods of labeling nucleic acids, such
as fragments of genomic DNA, with unique sequence tags, which may
include "mosaic tags," prior to amplification and sequencing. Such
sequence tags are useful for identifying amplification and
sequencing errors. Mosaic tags minimize sequencing and
amplification artifacts due to inappropriate annealing, priming,
hairpin formation, or the like, that may occur with completely
random sequence tags of the prior art. In one aspect, mosaic tags
are sequence tags that comprise alternating constant regions and
variable regions, wherein each constant region has a position in
the mosaic tag and comprises a predetermined sequence of
nucleotides and each variable region has a position in the mosaic
tag and comprises a predetermined number of randomly selected
nucleotides. By way of illustration, a 22-mer mosaic tag (SEQ ID
NO: 4) may have the following form:
Nucleotide Position:
TABLE-US-00001 ##STR00001##
[0027] Region Position
[0028] There are nine constant and variable regions, with regions 1
(nucleotides 1-3), 3 (nucleotide 9), 5 (nucleotides 12-14), 7
(nucleotides 18-19) and 9 (nucleotides 21-22) being variable
(double underlined nucleotides) and regions 2 (nucleotides 4-8), 4
(nucleotides 10-11), 6 (nucleotides 15-17), and 8 (nucleotide 20)
being constant. N represents a randomly selected nucleotide from
the set of A, C, G or T; thus, the number of mosaic tags of this
example is 4.sup.11=4,194,304 tags. b represents a predetermined
nucleotide at the indicated position. In some embodiments, the
sequence of b's, "***bbbbb*bb***bbb**b**", is selected to minimize
the likelihood of having a perfect match in a genome of the
organism making up the sample.
[0029] In one aspect, for mosaic tags of a particular embodiment of
the method of the invention, all constant regions with the same
position have the same length and all variable regions with the
same position have the same length. This allows mosaic tags to be
synthesized using partial combinatorial synthesis with conventional
chemistries and instruments.
[0030] In one aspect, mosaic tags comprise from 10 to 100
nucleotides, or from 12 to 80 nucleotides, or from 15 to 60
nucleotides. In some embodiments, mosaic tags comprise at least
eight nucleotide positions with randomly selected nucleotides; in
other embodiments, whenever mosaic tags have a length of at least
15 nucleotides, they comprise at least 12 nucleotide positions with
randomly selected nucleotides. In another aspect, no variable
region within a mosaic tag may have a length that is greater than
seven nucleotides.
[0031] In another aspect, mosaic tags may be used in the following
steps: (i) preparing DNA templates from nucleic acids in a sample;
(ii) labeling by sampling the DNA templates to form a multiplicity
tag-template conjugates, wherein substantially every DNA template
of a tag-template conjugate has a unique mosaic tag comprising
alternating constant regions and variable regions, each constant
region having a position in the mosaic tag and a length of from 1
to 10 nucleotides of a predetermined sequence and each variable
region having a position in the mosaic tag and a length of from 1
to 10 randomly selected nucleotides, such that constant regions
having the same positions have the same lengths and variable region
having the same positions have the same lengths; (iii) amplifying
the multiplicity of tag-template conjugates; (iv) generating a
plurality of sequence reads for each of the amplified tag-template
conjugates; and (v) determining a nucleotide sequence of each of
the nucleic acids by determining a consensus nucleotide at each
nucleotide position of each plurality of sequence reads having
identical mosaic tags. In another aspect, mosaic tags may be used
in the following steps: (a) preparing single stranded DNA templates
from nucleic acids in a sample; (b) labeling by sampling the single
stranded DNA templates to form tag-template conjugates, wherein
substantially every single stranded DNA template of a tag-template
conjugate has a unique sequence tag (that is, a mosaic tag) having
a length of at least 15 nucleotides and having the following form:
[0032] [(N.sub.1N.sub.2 . . . . N.sub.Kj)(b.sub.1b.sub.2 . . .
b.sub.Lj)].sub.M wherein each N.sub.i, for i=1, 2, . . . K.sub.j,
is a nucleotide randomly selected from the group consisting of A.
C, G and T; K.sub.j is an integer in the range of from 1 to 10 for
each j less than or equal to M (that is, regions N.sub.1N.sub.2 . .
. N.sub.Kj are variable regions), each b.sub.L, for i=1, 2, . . .
L.sub.j, is a nucleotide; L.sub.j is an integer in the range of
from 1 to 10 for each j less than or equal to M; such that every
sequence tag (i) has the same Kj for every j and (ii) has the same
sequences b.sub.1b.sub.2, . . . b.sub.Lj for every j (that is,
regions b.sub.1b.sub.2, . . . b.sub.Lj are constant regions); and M
is an integer greater than or equal to 2; (c) amplifying the
tag-template conjugates; (d) generating a plurality of sequence
reads for each of the amplified tag-template conjugates; and (e)
determining a nucleotide sequence of each of the nucleic acids by
determining a consensus nucleotide at each nucleotide position of
each plurality of sequence reads having identical sequence tags. In
some embodiments, the plurality of sequence reads is at least
10.sup.4; in other embodiments, the plurality of sequence reads is
at least 10.sup.5: in still other embodiments, the plurality of
sequence reads is at least 10.sup.6. In some embodiments, the total
length of the above sequence tag is in the range of from 15 to 80
nucleotides.
[0033] A variety of different attachment reactions may be used to
attach unique tags to substantially every clonotype in a sample. In
one embodiment, such attachment is accomplished by combining a
sample containing recombined nucleic acid molecules (which, in
turn, comprise clonotype sequences) with a population or library of
sequence tags so that members of the two populations of molecules
can randomly combine and become associated or linked, e.g.
covalently. In such tag attachment reactions, clonotype sequences
comprise linear single or double stranded polynucleotides and
sequence tags are carried by reagent such as amplification primers,
such as PCR primers, ligation adaptors, circularizable probes,
plasmids, or the like. Several such reagents capable of carrying
sequence tag populations are disclosed in Macevicz, U.S. Pat. No.
8,137,936; Faham et al, U.S. Pat. No. 7,862,999: Landegren et al.
U.S. Pat. No. 8,053,188: Unran and Deugau, Gene, 145: 163-169
(1994): Church, U.S. Pat. No. 5,149,625; and the like, which are
incorporated herein by reference.
[0034] FIGS. 1A and 1B illustrate an attachment reaction comprising
a PCR in which a population of sequence tags (T.sub.1, T.sub.2,
T.sub.3 . . . T.sub.j, T.sub.j+1, . . . T.sub.k, T.sub.k+1, . . .
T.sub.n-1, T.sub.n) is incorporated into primers (100). The
population of sequence tags has a much greater size than that of
recombined nucleic acid molecules (102). The sequence tags are
attached to the recombined nucleic acid molecules by annealing the
primers to the nucleic acid molecules and extending the primers
with a DNA polymerase in the first cycle of a PCR. The figure
depicts how the recombined nucleic acid molecules select, or
sample, a small fraction of the total population of sequence tags
by randomly annealing to the primers by way of their common primer
binding regions (104), for example, in V region (108). Since the
primers (an therefore sequence tags) combine with the recombined
nucleic acid sequence molecules randomly, there is a small
possibility that the same sequence tag may be attached to different
nucleic acid molecules; however, if the population of sequence tags
is large as taught herein, then such possibility will be negligibly
small so that substantially every recombined nucleic acid molecule
will have a unique sequence tag attached. The other primer (106) of
the forward and reverse primer pair anneals to C region (110) so
that after multiple cycles of annealing, extending and melting,
amplicon (112) is formed, thereby attaching unique sequence tags to
the V(D)J regions comprising the clonotypes of population (102).
That is, amplicon (112) comprises the tag-molecule conjugates from
the attachment reaction.
[0035] Such immune molecules typically form an immune repertoire
which comprises a very large set of very similar polynucleotides
(e.g. >1000, but more usually from 100,000 to 1,000,000, or
more) which are relatively short in length (e.g. usually less than
300 bp). In one aspect of the invention, the inventors recognized
and appreciated that these characteristics permitted the use of
highly dissimilar sequence tags to efficiently compare sequence
reads of highly similar clonotypes to determine whether they are
derived from the same original sequence or not.
[0036] The complexity of immune repertoires is well-known, e.g.
Arstila et al, Science, 286: 958-91 (1999) and Warren et al (cited
above). FIG. 1C illustrates diagrammatically a typical transcript
of an IgH molecule (120) from which a clonotype profile is derived
in accordance with some embodiments of the invention. Sources of
natural sequence variability include modular recombination of the
C, D, J and V segments from large sets carried by the genome,
nucleotide additions and deletions to the ends of the D segment to
produce the so-called "NDN" regions, and somatic hypermutation
where substitutions are made randomly over the length of transcript
(122) at a relative frequency roughly as indicated by curve (128).
In one aspect of the invention, complex populations of such IgH and
TCR transcripts are amplified and sequenced. In one aspect one or
both operations for IgH molecules are carried out by using
redundant primers annealing to different sites in the V regions
(described more fully below). This is particularly advantageous
where a sequencing chemistry is employed that has a relatively high
error rate or where such sequence variability is difficult or
impossible to know beforehand. In the latter case, primer extension
for amplification or generation of sequence reads takes place even
if one or more primer binding sites are inoperable, or
substantially inoperable, because of mismatches caused (for
example) by one or more somatic mutations. Starting from promoter P
(122) relative frequency shown by curve (128) climbs through leader
region (124) to a maximum over the V(D)J region (126) of the
transcript after which it drop to near zero. In one aspect of the
invention, a segment of recombined B cell nucleic acid is amplified
by a PCR with a plurality of forward primers or a plurality of
reverse primers to generate a nested set of templates, e.g. as
disclosed in Faham and Willis, U.S. patent publication
2011/0207134. Templates from such a set may be further amplified on
a surface to form separate amplicons (e.g. by bridge PCR using a
cBot instrument, Illumina, San Diego, Calif.). Templates from the
same nested set may be associated with one another by sequence
reads generated at their common ends. Nested sets of templates
allow a sequencing chemistry with relative high error rates to be
used to analyze longer sequences than otherwise would be possible,
while at the same time maintaining high average quality scores over
the entire length of the sequence. The nested sets also ensure that
at least one sequence read is obtained from a V region even if it
has been subjected to somatic hypermutation.
[0037] Somatic mutations in IgH molecules add a layer of difficulty
in reconstructing clonotypes from sequence read data, because of
the difficulty in determining whether a base change is due to
sequencing or amplification error or is due to a natural mutation
process. The use of sequence tags in accordance with the invention
greatly mitigates such difficulties because every nucleic acid
encoding an IgH will receive a distinct and unique sequence tag. As
illustrated in FIG. 3, sequence reads (300) pursuant to the
invention each comprise a copy of a sequence tag (302) and a copy
of a clonotype (304) (SEQ ID NO: 1). All sequence reads having the
same sequence lag are assembled so that the nucleotides of each
position in the clonotype portion can be compared (e.g., SEQ ID NO:
2 and SEQ ID NO: 3). Thus, even if IgH-encoding sequences differ by
no more than a single base, they will receive a distinct sequence
tag, so that closely related IgH-encoding nucleic acids in a sample
are not compared with one another in the clonotype determination
process. As mentioned above, errors in the sequence tags are not
significant because the sequences of the sequence tags associated
with clonotypes are so far apart in sequence space that a huge
number of base changes could be sustained without one sequence tag
becoming close in sequence space to any other sequence tag.
[0038] Constructing clonotypes from sequence read data depends in
part on the sequencing method used to generate such data, as the
different methods have different expected read lengths and data
quality. In one approach, a Solexa sequencer is employed to
generate sequence read data for analysis. In one embodiment, a
sample is obtained that provides at least 0.5-1.0.times.10.sup.6
lymphocytes to produce at least 1 million template molecules, which
after optional amplification may produce a corresponding one
million or more clonal populations of template molecules (or
clusters). For most high throughput sequencing approaches,
including the Solexa approach, such over sampling at the cluster
level is desirable so that each template sequence is determined
with a large degree of redundancy to increase the accuracy of
sequence determination. For Solexa-based implementations,
preferably the sequence of each independent template is determined
10 times or more. For other sequencing approaches with different
expected read lengths and data quality, different levels of
redundancy may be used for comparable accuracy of sequence
determination. Those of ordinary skill in the art recognize that
the above parameters, e.g. sample size, redundancy, and the like,
are design choices related to particular applications.
[0039] FIGS. 2A-2C illustrate exemplary steps of attaching unique
sequence tags to recombined nucleic acid molecules in a two stage
PCR. Population of recombined nucleic acid molecules (250) from a
sample containing T-cells or B-cells are combined in a PCR mixture
with forward and reverse primers (202) and (262). Primers (262)
each comprise three regions: target annealing region (263) (which
in this illustration is V region (206)); sequence tag (264); and
primer binding region (265) for the second stage of the two-stage
PCR. In this illustration, primers (262) comprise a mixture of
target annealing regions to account for the diversity of V region
sequence. Thus, every different primer is prepared with a sequence
tag region. Alternatively, the sequence tag element may be attached
to C region primer (202) along with a primer binding region for the
second PCR stage. As noted, recombined nucleic acid molecules (250)
comprise constant, or C, region (203), J region (210), D region
(208), and V region (206), which may represent a V(D)J segment
encoding a CDR3 region of either a TCR or immunoglobulin. After a
few cycles, for example, 4 to 10, first stage amplicon (266) is
produced with each member polynucleotide including sequence tag
(270). In the second stage PCR, polynucleotides of amplicon (266)
are reamplified with new forward and reverse primers P5 (222) and
P7 (220) which add further primer binding sites (224) and (223) for
cluster formation using bridge PCR in a Solexa/Illumina sequencer.
Primer P7 also include a secondary sequence tag (221) for optional
multiplexing of samples in a single sequencing run. After the
secondary PCR amplicon (280) is produced with embedded P5 and P7
sequences by which bridge PCR may be carried out.
Clonotype Determination from Sequence Data
[0040] In accordance with one aspect of the invention, clonotypes
of a sample are determined by first grouping sequence reads based
on their sequence tags. Such grouping may be accomplished by
conventional sequence alignment methods. Guidance for selecting
alignment methods is available in Batzoglou. Briefings in
Bioinformatics, 6: 6-22 (2005), which is incorporated by reference.
After sequence reads are assembled in groups corresponding to
unique sequence tags, then the sequences of the associated
clonotypes may be analyzed to determine the sequence of the
clonotype from the sample. FIG. 3 illustrates an exemplary
alignment and method from determining the sequence (SEQ ID NO: 1)
of a clonotype associated with a unique sequence tag. In this
example, eleven sequence reads are aligned by way of their
respective sequence tags (302) after which nucleotides at each
position of the clonotype portions of the sequence reads, indicated
as 1, 2, 3, 4, . . . n, are compared. For example, nucleotides at
position 6 (306) are t, t, g, t, t, t, t, t, t, t, c, t; that is,
nine base calls are t's, one is "g" (308) and one is "c" (310) (SEQ
ID NO: 2 and SEQ ID NO: 3, respectively). In one embodiment, the
correct base call of the clonotype sequence at a position is
whatever the identity of the majority base is. In the example of
position 6 (306), the base call is "t", because it is the
nucleotide in the majority of sequence reads at that position. In
other embodiments, other factors may be taken into account to
determine a correct base call for a clonotype sequence, such as
quality scores of the base calls of the sequence reads, identities
of adjacent bases, or the like.
[0041] Once clonotypes are determined as described above, a
clonotype profile comprising the abundances or frequencies of each
different clonotype of a sample may be assembled.
Homopolymer Determination in Sequence-by-Synthesis
Methodologies
[0042] In accordance with an aspect of the invention, sequence tags
may be used for determining the number of nucleotides in a
homopolymer region from signals generated in a
sequencing-by-synthesis (SBS) method. That is, whenever a target
polynucleotide has been replicated and multiple replicates
subjected to separate SBS reactions, each replicate may be labeled
with the same sequence tag so that SBS signals from base
incorporations in similar regions of the separate replicates may be
analyzed by linking the different incorporation signals through the
common sequence tags. SBS methodologies have a wide variety of
implementations, e.g. Fuller et al, Nature Biotechnology, 27:
1013-1023 (2009); Ronaghi et al, Science, 281: 363-365 (1998);
Margulies et al, Nature, 437: 376-380 (2005): Rothberg et al.
Nature, 475: 348-352 (2011); Kumar et al, Scientific Reports, 2:
684 (2012); Sims et al, Nature Methods, 8: 575-580 (2011); Pourmand
et al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006); Seo et al,
Proc. Natl. Acad. Sci. 102: 5926-5931 (2005): and the like.
Commercial DNA sequencers using SBS include 454 GS sequencers (454
Life Sciences), PGM and Proton Ion Torrent sequencers (Life
Technologies), PyroMark sequencers (Qiagen), and the like. A common
feature of many variants of the SBS methodologies is the generation
of a sequence of signals ("incorporation signals") each of which is
proportional to (or at least monotonically related to) the number
of nucleotides incorporated into an extended nucleotide chain in a
template-driven synthesis reaction. A common challenge with such
variants of SBS is determining the number of nucleotides
incorporated in homopolymeric regions of a template when the length
of the homopolymeric region exceeds 5-6 nucleotides. In one aspect
of the invention, this problem is addressed by the use of sequence
tags. As above, sequences of such sequence tags in this embodiment
are sufficiently far apart from one another that they are readily
related to their progenitor, or parent, sequences even after
incurring multiple amplification and/or sequencing errors. In this
manner, they are distinct from tags frequently employed in
sequencing, such as tag for calibrating base-calling software (e.g.
so-called "key" sequences), designating sample or specimen origin,
reducing carry-over contamination, or the like. In some
embodiments, templates are labeled by sampling using mosaic tags so
that the sequence tags themselves may be selected without
homopolymer regions greater than the length of the longest variable
region of the tag. In some embodiments, mosaic tags are used with
variable regions not longer than 5 nucleotides; and in other
embodiments, mosaic tags are used with variable regions not longer
than 4 nucleotides. Alternatively, sequence tags suitable for SBS
may be constructed as disclosed in Brenner et al, U.S. Pat. No.
7,537,897, which is hereby incorporated by reference.
[0043] FIG. 4A illustrates one variant of the SBS methodology that
comprises cycles of nucleotide addition and washing, with signal
generation typical going on during or after incorporation of an
added nucleotide. In other words, the illustrated variant comprises
cycles of sequentially adding each of the different nucleoside
triphosphates and washing, such as in the following order: (a)
adding dATP to a growing template-bound chain and washing; (b)
adding dGTP to the growing template-bound chain and washing; (c)
adding dCTP to the growing template-bound chain and washing; and
(d) adding dTTP to the growing template-bound chain and washing.
Many additional embodiments and variations of such cycles are
possible, e.g. Schultz et al, U.S. patent publication 2012/0035062,
and come within the scope of sequencing by synthesis. Typically the
extensions are carried out with a nucleic acid polymerase, such as
a DNA polymerase. Returning to FIG. 4A, showing one variant of SBS,
single stranded template (400) having sequence region (402)
adjacent to primer binding site (404) is anchored to solid phase
support (408), such as a SNAPP in a microwell of an Ion Torrent
semiconductor sequencing chip, e.g. Mobile et al, U.S. patent
publication 2010/0300895; Hinz et al, U.S. patent publication
2011/0195253, which are incorporated herein by reference. Anchored
template (400) is combined with sequencing primer (406) and
polymerase (410) under conditions that permit the annealing of
sequencing primer (406) to primer binding site (404) and the
extension of sequencing primer (406) by polymerase (410) upon
addition of nucleoside triphosphates. In the variant of FIG. 4A,
the complex comprising template (400), sequencing primer (406) and
polymerase (410) is exposed separately and sequentially to flows of
dATP, dGTP, dCTP and dTTP. After exposure to dATP flow (412),
sequencing primer (406) is extended by a single dA (413). After
washing (414) to remove or destroy any residual dATP, dGTP flow
(416) is introduced, so that polymerase (410) incorporates a dGTP
to extend sequencing primer (406) by a single dG (415). After
washing (418), dCTP flow (420) is introduced, however, because the
next base in the template is not complementary to dC, there is no
nucleotide incorporation and no extension of sequencing primer
(406). The cycle is completed with washing step (422) and dTTP flow
(424).
[0044] During or after each of the incorporation events in this and
other SBS variants, an incorporation signal is generated, which may
vary widely in character and may comprise more than one physical or
chemical manifestations at the same time, e.g. optical and/or
electrical. Exemplary incorporation signals include, but are not
limited to changes in pH, fluorescence, chemiluminescence,
resistance, and the like. FIG. 4B illustrates typical incorporation
signals recorded (426, 428, 430) during or after each of the above
flows of dATP, dGTP and dCTP of FIG. 4A, respectively. Such data
may be analyzed to give a number for each peak that is proportional
to or monotonically related to, the number of nucleotides that are
added to primer (406), or extensions thereof. The number obtained
in such an analysis may vary according to particular embodiments,
for example, such a number may be a function of the area of such
peaks, peak heights, peak width at half maximum, or like functions
known in the art. FIGS. 4C and 4D illustrate the SBS process in the
presence of homopolymer region (432) and the use of sequence tags
for determining incorporation signals. Supports (436, 438, 440)
correspond to separate SBS reactions 1, 2 and K, respectively,
which have replicates of the same sequence tag-template conjugates
attached (SEQ ID NO: 5). Primers 406, 406' and 406'' are extended
in separate reactions by separate polymerases 410, 410' and 410'',
respectively. Such separate reactions are implemented in separate
reaction confinement regions which, in turn, may have a variety of
forms, such as arrays of microwells of various scales, e.g.
Rothberg et al, Nature, 475:348-352 (2011); Learnon et al, U.S.
Pat. No. 8,158,359; Rothberg et al, U.S. Pat. No. 8,313,625; which
are each incorporated herein by reference. Reaction confinement
regions may also be defined by clusters of template nucleic acids
on beads or surfaces or within gels, e.g. as produced by bridge
PCR, disclosed in Boles et al, U.S. Pat. No. 6,300,070;
Balasubramanian et al. U.S. Pat. No. 6,787,308, both of which are
incorporated by reference. In accordance with some embodiments of
SBS methodology, different dNTP flows are introduced sequentially
to each of the reactions. Typically, but not necessarily, each
different reaction would be exposed to the same flow at
approximately the same time, e.g. as would be the case for SBS
reactions taking place in different microwells of an Ion Torrent
semiconductor sequencing chip used in conjunction with an
appropriate flow system, e.g. Nobile et at (cited above): Davey et
al, U.S. patent publication 2012/0143531, the latter of which is
incorporated herein by reference. During or after each dNTP flow,
incorporation signals (i.sub.1, i.sub.2, . . . i.sub.k) are
obtained. FIG. 4C illustrates the incorporation signals for the
second dATP flow (442). For homopolymers exceeding 5-6 bases, the
contribution of the last-to-be-incorporated nucleotides to the
total signal becomes less and less, so that it becomes difficult to
distinguish signal from an incorporated nucleotide from noise. In
accordance with the invention, incorporation signals from multiple
replicate templates may be analyzed together because they can be
identified by their common sequence tags. As discussed above, the
sequence tags are selected (such as, by a labeling by sampling
process) so that even with multiple amplification or sequencing
errors present, sequence tags of each tag-polynucleotide conjugate
and its replicates are distinguishable from sequence tags of any
other tag-polynucleotide conjugate and each of its replicates.
[0045] FIG. 4D shows multiple (1, 2, . . . K) sequences of
magnitudes of incorporation signals (450), wherein each sequence is
associated with common sequence tag (452) and template
TCGGGGGGGACTT (SEQ ID NO: 5). The form of the illustrated data set
is similar to that illustrated in FIG. 3, which illustrates how
columns of base measurements could be used to determine a final
base call. FIG. 4D illustrates how columns of incorporation signals
can be used to make a final determination of an nucleotide
incorporation number. Such determinations may be made after a
separate model is used to extract nucleotide incorporate estimates
from incorporation signals from individual reactions, or the
determinations may be made by applying a model to all or a
plurality of the incorporation signals from tag-polynucleotide
conjugates with the same parent tag. As noted above, the numbers
shown in FIG. 4D are the values of a function of the data sets
illustrated by peaks such as (426, 428 and 430) (which show the
rate of generation of an incorporation signal versus time and whose
integration, or area, is one measure of nucleotide incorporation).
The columns of FIG. 4D, labeled "f1a, f1g, f1c, f1t, f2a, f2g" and
so on, represent cycles of dNTP flows, wherein the first cycle
(f1a, f1g, f1c, f1t) comprises sequential introduction of dATP,
dGTP, dCTP and dTTP to the SBS reactions, and so on.
[0046] As mentioned above, this aspect of the invention may be
implement by a method having the steps: (a) attaching a sequence
tag to each of the one or more polynucleotides to form
tag-polynucleotide conjugates, wherein substantially every
polynucleotide of the tag-polynucleotide conjugates has a unique
sequence tag; (b) amplifying the tag-polynucleotide conjugates; (c)
sequencing by synthesis amplified tag-polynucleotide conjugates,
wherein sequencing by synthesis comprises at least one dNTP flow;
and (d) determining for each tag-polynucleotide having the same
sequence tag a number of nucleotide incorporations for each dNTP
flow as a function of measured incorporation signals for each such
dNTP flow. Typically amplified tag-polynucleotide conjugates
include flanking sequences to assist in implementing the various
steps. For example, tag-polynucleotide conjugates may include one
or more flanking primer binding sites at each end to permit such
operations as secondary amplification, sequencing primer annealing
and extension, hybridization-based capture, and the like. As
mentioned above, dNTP flows may be implemented by a wide variety of
fluid delivery apparatus and control systems, such as those
implemented on most commercially available DNA sequencing systems,
e.g. Schultz et al, U.S. patent publication 2012/0073667, which is
incorporated herein by reference. A dNTP flow is a movement of
dNTP-containing reagent through a reaction site. Preferably, dNTP
flows are implemented so that there is a sharp fluid boundary
between successive flows. In some embodiments, washing steps may be
implemented with reagents substantially identical to
dNTP-containing reagent, except for the presence of a dNTP. In some
embodiments, reagent for implementing washing steps may include
agents to destroy or denature dNTPs of a prior flow, e.g. apyrase.
In most embodiments, a dNTP flow contains all the reagents, salt
levels, pH and the like, sufficient for a polymerase in a complex
to extend a sequencing primer by incorporation of dNTPs of the flow
in a template-driven reaction along the tag-polynucleotide
conjugate. As mentioned above, a wide variety of functions or
algorithms may be used to model signals (e.g. incorporation signal
values) from separate reactions related by having
tag-polynucleotide conjugates with the same sequence tag. In some
embodiments, the function of incorporation signals from
tag-polynucleotide conjugates having the same sequence tag is the
whole number closest to an average of such incorporation signals
measured from the separate SBS reactions. The average may be an
arithmetic average or a weighted average, e.g. the latter of which
may depend on characteristics of a particular sequencing system
used, such as the particular sequencing chemistry used, whether
labels are used, what type of signal is generated (e.g. optical,
resistive pulse, pH, etc.), the location of an SBS reaction site in
a dNTP flow, and the like. Guidance for alternative models that
relate signals from an SBS reaction and nucleotides incorporated
are disclosed in Rearick, U.S. patent publication 2012/0172241; and
Hubbell, U.S. patent publication 2012/0173158, both of which are
incorporated herein by reference.
[0047] As mentioned above, sequencing by synthesis may be
implemented by a wide variety of process steps and reagents. In
some embodiments, sequencing by synthesis may be implemented with
the following steps: (a) forming for each of a plurality of
tag-polynucleotide conjugates a complex comprising a sequencing
primer, a nucleic acid polymerase, and a tag-polynucleotide
conjugate under conditions that permit annealing of the sequencing
primer to such tag-polynucleotide conjugate and extension of such
sequencing primer along the tag-polynucleotide conjugate in the
presence of nucleoside triphosphates by the nucleic acid
polymerase; (b) introducing a dNTP to the complexes by a dNTP flow;
(c) measuring incorporation signals from each complex; (d) washing
the complexes; and (e) repeating steps (b) through (d). In some
embodiments, after tag-polynucleotide conjugates are amplified, a
sample of the resulting amplicon is used in the sequencing by
synthesis steps. In some embodiments, such sample comprises at
least a number of tag-polynucleotide conjugates greater than
10.sup.2; in other embodiments, such sample comprise a number of
tag-polynucleotide conjugates in the range of from 10.sup.2 to
10.sup.15, or in other embodiments, in the range of from 10.sup.2
to 10.sup.12, or in other embodiments, in the range of from
10.sup.2 to 10.sup.9. In other embodiments, such sample is
sufficiently large so that all polynucleotides having a frequency
of 0.001 or higher are present with a probability of ninety-nine
percent; or all polynucleotides having a frequency of 0.0001 or
higher are present with a probability of ninety-nine percent.
[0048] dNTPs employed in this aspect of the invention may be
unblocked dNTPs or extension-block dNTPs. In the case of the former
(unblocked dNTPs), during a single dNTP flow, if a template
contains a homopolymeric region, then a number incorporations may
occur that is equal to the homopolymer length. (There may be
factors that cause fewer incorporations to be made (e.g. a
polymerase with low processivity), but with unblocked dNTPs it is
possible for such number to equal homopolymer length). For example,
the Ion Torrent sequencing by synthesis chemistry employs unblocked
dNTPs. In the case of extension-blocked dNTPs, only a single dNTP
incorporation can take place for each flow, usually because of the
presence of a chemical blocking group on an incorporated
nucleotide. Thus, cycles of dNTP incorporation further include a
de-blocking step in order to regenerate an extendable end on the
extended sequencing primer. For example, the Illumina sequencing by
synthesis chemistry employs extension-blocked dNTPs, e.g. Bentley
et al (cited below). In some embodiments, a de-blocking step may be
implemented as part of a washing step.
Use of Sequence Tags to Detect Carry Over Contamination
[0049] Carry over contamination is a significant problem with
techniques that include amplification of nucleic acids, e.g. Borst
et al, Ear. J. Clin. Microbiol. Infect. Dis. 23(4): 289-299 (2004);
Aslanzadeh, Ann. Clin. Lab. Sci. 34(4): 389-396 (2004); and the
like. Such contamination arises when traces of nucleic acid
extraneous to a sample are unintentionally amplified in an assay of
the sample and effect or impact a measured result. In a worse case,
carry over contamination in a medical sample from a patient can
result in a false positive interpretation of an assay result. The
extraneous nucleic acid may come from a source unrelated to a
particular patient; for example, it may come from the sample of
another patient. Or, the extraneous nucleic acid may come from a
source related to a patient; for example, it may come from a
different sample from the same patient handled in the same
laboratory in the past or from an assay reaction on a different
sample from the same patient which was processed in the same
laboratory in the past.
[0050] Carry over contamination is especially challenging in a
clinical setting when measuring highly complex populations of
related nucleic acids, such as populations of recombined nucleic
acids encoding immune molecules, such as T-cell receptors or
immunoglobulins. The challenge arises because it is difficult to
determine whether a sequence read or clonotype is part of the
genuine diversity of an intended sample or whether they originate
from an extraneous source of nucleic acid, such as another
patient's sample or a prior sample of the same patient, which are
being processed in the same king of assay in the same laboratory.
In one aspect of the invention, such carry over contamination may
be detected by using sequence tags not only to determine clonotypes
from sequence reads but also to determine whether a sequence tag
originated in the current sample or from another sample. This is
accomplished by maintaining a record of sequence tags determined
from each patient sample, then whenever a subsequent measurement is
made the sequence tags of the current measurement are compared to
those of prior measurements. Such records of sequence tags
associated with clonotypes are conveniently maintained as
electronic records on mass storage devices because of the large
number of tag from each measurement and the ease of searching and
comparing electronic records using conventional algorithms. If a
match is found then the most likely cause is carry over
contamination, provided that the populations of sequence tags
employed in the measurements are sufficiently large. The same
exemplary ratios of the size of sequence tag population to a
clonotype population for labeling by sampling discussed above are
applicable for detecting carry over contamination. In one
embodiment, such ratio is 100:1 or greater.
Samples
[0051] Clonotype profiles are obtained from samples of immune
cells, which are present in a wide variety of tissues. Immune cells
of interest include T-cells and/or B-cells. T-cells (T lymphocytes)
include, for example, cells that express T cell receptors (TCRs).
B-cells (B lymphocytes) include, for example, cells that express B
cell receptors (BCRs). T-cells include helper T cells (effector T
cells or Th cells), cytotoxic T cells (CTLs), memory T cells, and
regulatory T cells, which may be distinguished by cell surface
markers. In one aspect a sample of T cells includes at least 1,000
T cells; but more typically, a sample includes at least 10,000 T
cells, and more typically, at least 100,000 T cells. In another
aspect, a sample includes a number of T cells in the range of from
1000 to 1,000,000 cells. A sample of immune cells may also comprise
B cells. B-cells include, for example, plasma B cells, memory B
cells, B1 cells, B2 cells, marginal-zone B cells, and follicular B
cells. B-cells can express immunoglobulins (also referred to as
antibodies or B cell receptors). As above, in one aspect a sample
of B cells includes at least 1,000 B cells; but more typically, a
sample includes at least 10,000 B cells, and more typically, at
least 100,000 B cells. In another aspect, a sample includes a
number of B cells in the range of from 1000 to 1,000,000 B
cells.
[0052] Samples (sometimes referred to as "tissue samples") used in
the methods of the invention can come from a variety of tissues,
including, for example, tumor tissue, blood and blood plasma, lymph
fluid, cerebrospinal fluid surrounding the brain and the spinal
cord, synovial fluid surrounding bone joints, and the like. In one
embodiment, the sample is a blood sample. The blood sample can be
about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0,
2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mL. The sample can be a tumor
biopsy. The biopsy can be from, for example, from a tumor of the
brain, liver, lung, heart, colon, kidney, or bone marrow. Any
biopsy technique used by those skilled in the an can be used for
isolating a sample from a subject. For example, a biopsy can be an
open biopsy, in which general anesthesia is used. The biopsy can be
a closed biopsy, in which a smaller cut is made than in an open
biopsy. The biopsy can be a core or incisional biopsy, in which
part of the tissue is removed. The biopsy can be an excisional
biopsy, in which attempts to remove an entire lesion are made. The
biopsy can be a fine needle aspiration biopsy, in which a sample of
tissue or fluid is removed with a needle.
[0053] A sample or tissue sample includes nucleic acid, for
example, DNA (e.g., genomic DNA) or RNA (e.g., messenger RNA). The
nucleic acid can be cell-free DNA or RNA, e.g. extracted from the
circulatory system, Vlassov et al Curr. Mol. Med., 10: 142-165
(2010): Swarup et al, FEBS Lett., 581: 795-799 (2007). In the
methods of the invention, the amount of RNA or DNA from a subject
that can be analyzed includes varies widely. For example. DNA or
RNA of a single cell may be all that is required for a calibration
test (i.e. an initial measurement to determine a correlating
clonotype for a disease). For generating a clonotype profile,
sufficient nucleic acid must be in a sample to obtain a useful
representation of an individual's immune receptor repertoire. More
particularly, for generating a clonotype profile from genomic DNA
at least 1 ng of total DNA from T cells or B cells (i.e. about 300
diploid genome equivalents) is extracted from a sample: in another
embodiment, at least 2 ng of total DNA (i.e. about 600 diploid
genome equivalents) is extracted from a sample: and in another
embodiment, at least 3 ng of total DNA (i.e. about 900 diploid
genome equivalents) is extracted from a sample. One of ordinary
skill would recognize that as the fraction of lymphocytes in a
sample decreases, the foregoing minimal amounts of DNA must
increase in order to generate a clonotype profile containing more
than about 1000 independent clonotypes. For generating a clonotype
profile from RNA, in one embodiment, a sufficient amount of RNA is
extracted so that at least 1000 transcripts are obtained which
encode distinct TCRs, BCRs, or fragments thereof. The amount of RNA
that corresponds to this limit varies widely from sample to sample
depending on the fraction of lymphocytes in a sample, developmental
stage of the lymphocytes, and the like. In one embodiment, at least
100 ng of RNA is extracted from a tissue sample containing B cells
and/or T cells for the generating of a clonotype profile: in
another one embodiment, at least 500 ng of RNA is extracted from a
tissue sample containing B cells and/or T cells for the generating
of a clonotype profile. RNA used in methods of the invention may be
either total RNA extracted from a tissue sample or polyA RNA
extracted directly from a tissue sample or from total RNA extracted
from a tissue sample. The above nucleic acid extractions may be
carried out using commercially available kits, e.g. from Invitrogen
(Carlsbad, Calif.). Qiagen (San Diego, Calif.), or like vendors.
Guidance for extracting RNA is found in Liedtke et al, PCR Methods
and Applications, 4: 185-187 (1994); and like references.
[0054] As discussed more fully below (Definitions), a sample
containing lymphocytes is sufficiently large so that substantially
every T cell or B cell with a distinct clonotype is represented
therein, thereby forming a repertoire (as the term is used herein).
In one embodiment, a sample is taken that contains with a
probability of ninety-nine percent every clonotype of a population
present at a frequency of 0.001 percent or greater. In another
embodiment, a sample is taken that contains with a probability of
ninety-nine percent every clonotype of a population present at a
frequency of 0.0001 percent or greater. In one embodiment, a sample
of B cells or T cells includes at least a half million cells, and
in another embodiment such sample includes at least one million
cells.
[0055] Whenever a source of material from which a sample is taken
is scarce, such as, clinical study samples, or the like, DNA from
the material may be amplified by a non-biasing technique, such as
whole genome amplification (WGA), multiple displacement
amplification (MDA); or like technique, e.g. Hawkins et al, Curr.
Opin. Biotech., 13: 65-67 (2002); Dean et al, Genome Research, 11:
1095-1099 (2001); Wang et al, Nucleic Acids Research, 32: e76
(2004): Hosono et al, Genome Research, 13: 954-964 (2003); and the
like.
[0056] Blood samples are of particular interest and may be obtained
using conventional techniques, e.g. Innis et al, editors, PCR
Protocols (Academic Press, 1990); or the like. For example, white
blood cells may be separated from blood samples using convention
techniques, e.g. RosetteSep kit (Stem Cell Technologies, Vancouver.
Canada). Likewise, other fractions of whole blood, such as
peripheral blood mononuclear cells (PBMCs) may be isolated for use
with methods of the invention using commercially available kits,
e.g. Miltenyi Biotec, Auburn, Calif.), or the like. Blood samples
may range in volume from 100 .mu.L to 10 mL; in one aspect, blood
sample volumes are in the range of from 200 100 .mu.L to 2 mL. DNA
and/or RNA may then be extracted from such blood sample using
conventional techniques for use in methods of the invention, e.g.
DNeasy Blood & Tissue Kit (Qiagen, Valencia, Calif.).
Optionally, subsets of white blood cells, e.g. lymphocytes, may be
further isolated using conventional techniques, e.g. fluorescently
activated cell sorting (FACS) (Becton Dickinson, San Jose, Calif.),
magnetically activated cell sorting (MACS) (Miltenyi Biotec,
Auburn, Calif.), or the like.
[0057] In some embodiments, recombined nucleic acids are present in
the DNA of each individual's adaptive immunity cells as well as
their associated RNA transcripts, so that either RNA or DNA can be
sequenced in the methods of the provided invention. A recombined
sequence from a T-cell or B-cell encoding a T cell receptor or
immunoglobulin molecule, or a portion thereof, is referred to as a
clonotype. The DNA or RNA can correspond to sequences from T-cell
receptor (TCR) genes or immunoglobulin (Ig) genes that encode
antibodies. For example, the DNA and RNA can correspond to
sequences encoding .alpha., .beta., .gamma., or .delta. chains of a
TCR. In a majority of T-cells, the TCR is a heterodimer consisting
of an .alpha.-chain and .beta.-chain. The TCR.alpha. chain is
generated by VJ recombination, and the .beta. chain receptor is
generated by V(D)J recombination. For the TCR.beta. chain, in
humans there are 48 V segments, 2 D segments, and 13 J segments.
Several bases may be deleted and others added (called N and P
nucleotides) at each of the two junctions. In a minority of
T-cells, the TCRs consist of .gamma. and .delta. delta chains. The
TCR chain is generated by VJ recombination, and the TCR .delta.
chain is generated by V(D)J recombination (Kenneth Murphy, Paul
Travers, and Mark Walport. Janeway's Immunology 7th edition,
Garland Science, 2007, which is herein incorporated by reference in
its entirety).
[0058] The DNA and RNA analyzed in the methods of the invention can
correspond to sequences encoding heavy chain immunoglobulins (IgH)
with constant regions (.alpha., .delta., .epsilon., .gamma., or
.mu.) or light chain immunoglobulins (IgK or IgL) with constant
regions .lamda. or .kappa.. Each antibody has two identical light
chains and two identical heavy chains. Each chain is composed of a
constant (C) and a variable region. For the heavy chain, the
variable region is composed of a variable (V), diversity (D), and
joining (J) segments. Several distinct sequences coding for each
type of these segments are present in the genome. A specific VDJ
recombination event occurs during the development of a B-cell,
marking that cell to generate a specific heavy chain. Diversity in
the light chain is generated in a similar fashion except that there
is no D region so there is only VJ recombination. Somatic mutation
often occurs close to the site of the recombination, causing the
addition or deletion of several nucleotides, further increasing the
diversity of heavy and light chains generated by B-cells. The
possible diversity of the antibodies generated by a B-cell is then
the product of the different heavy and light chains. The variable
regions of the heavy and light chains contribute to form the
antigen recognition (or binding) region or site. Added to this
diversity is a process of somatic hypermutation which can occur
after a specific response is mounted against some epitope.
[0059] In accordance with the invention, primers may be selected to
generate amplicons of subsets of recombined nucleic acids extracted
from lymphocytes. Such subsets may be referred to herein as
"somatically rearranged regions." Somatically rearranged regions
may comprise nucleic acids from developing or from fully developed
lymphocytes, where developing lymphocytes are cells in which
rearrangement of immune genes has not been completed to form
molecules having full V(D)J regions. Exemplary incomplete
somatically rearranged regions include incomplete IgH molecules
(such as, molecules containing only D-J regions), incomplete
TCR.delta. molecules (such as, molecules containing only D-J
regions), and inactive IgK (for example, comprising Kde-V
regions).
[0060] Adequate sampling of the cells is an important aspect of
interpreting the repertoire data, as described further below in the
definitions of "clonotype" and "repertoire." For example, starling
with 1,000 cells creates a minimum frequency that the assay is
sensitive to regardless of how many sequencing reads are obtained.
Therefore one aspect of this invention is the development of
methods to quantitate the number of input immune receptor
molecules. This has been implemented this for TCR.beta. and IgH
sequences. In either case the same set of primers are used that are
capable of amplifying all the different sequences. In order to
obtain an absolute number of copies, a real time PCR with the
multiplex of primers is performed along with a standard with a
known number of immune receptor copies. This real time PCR
measurement can be made from the amplification reaction that will
subsequently be sequenced or can be done on a separate aliquot of
the same sample. In the case of DNA, the absolute number of
rearranged immune receptor molecules can be readily converted to
number of cells (within 2 fold as some cells will have 2 rearranged
copies of the specific immune receptor assessed and others will
have one). In the case of cDNA the measured total number of
rearranged molecules in the real time sample can be extrapolated to
define the total number of these molecules used in another
amplification reaction of the same sample. In addition, this method
can be combined with a method to determine the total amount of RNA
to define the number of rearranged immune receptor molecules in a
unit amount (say 1 g) of RNA assuming a specific efficiency of cDNA
synthesis. If the total amount of cDNA is measured then the
efficiency of cDNA synthesis need not be considered. If the number
of cells is also known then the rearranged immune receptor copies
per cell can be computed. If the number of cells is not known, one
can estimate it from the total RNA as cells of specific type
usually generate comparable amount of RNA. Therefore from the
copies of rearranged immune receptor molecules per 1 .mu.g one can
estimate the number of these molecules per cell.
[0061] One disadvantage of doing a separate real time PCR from the
reaction that would be processed for sequencing is that there might
be inhibitory effects that are different in the real time PCR from
the other reaction as different enzymes, input DNA, and other
conditions may be utilized. Processing the products of the real
time PCR for sequencing would ameliorate this problem. However low
copy number using real time PCR can be due to either low number of
copies or to inhibitory effects, or other suboptimal conditions in
the reaction.
[0062] Known amounts of one or more internal standards to cDNA or
genomic DNA can be added to an assay reaction to determine absolute
quantities or concentrations of cDNA or genomic DNA samples of
unknown quantity. By counting the number of molecules of the
internal standard and comparing it to the rest of the sequences of
the same sample, one can estimate the number of rearranged immune
receptor molecules in the initial cDNA sample. (Such techniques for
molecular counting are well-known, e.g. Brenner et al, U.S. Pat.
No. 7,537,897, which is incorporated herein by reference).
Amplification of Nucleic Acid Populations
[0063] Amplicons of target populations of nucleic acids,
particularly recombined immune molecules, may be generated by a
variety of amplification techniques. In one aspect of the
invention, multiplex PCR is used to amplify members of a mixture of
recombined immune molecules, such as T cell receptors or portions
thereof or B cell receptors or portions thereof. Guidance for
carrying out multiplex PCRs of such immune molecules is found in
the following references, which are incorporated by reference:
Faham and Willis, U.S. Pat. No. 8,236,503; Morley, U.S. Pat. No.
5,296,351; Gorski, U.S. Pat. No. 5,837,447; Dau, U.S. Pat. No.
6,087,096; Von Dongen et al, U.S. patent publication 2006/0234234;
European patent publication EP 1544308B1; and the like. In some
embodiments of the invention, a step of generating a clonotype
profile include steps of (a) amplifying a portion of T cell
receptor genes and/or a portion of B cell receptor genes and (b)
sequencing nucleic acids of the resulting amplicon. As explained
elsewhere, the number of amplicon nucleic acids sequenced may vary
from application to application. For example, a clonotype profile
to determine whether a leukemia patient is still in remission will
be large so that the limit of detection of any tumor clones will be
very low. In some embodiments, the number of amplicon nucleic acids
sequenced is at least 1000; in other embodiments, the number of
amplicon nucleic acids sequenced is at least 10.sup.4; in other
embodiments, the number of amplicon nucleic acids sequenced is at
least 10.sup.5. Such a generating step may also include further
steps of coalescing sequence reads into clonotypes, enumerating or
tabulating clonotypes, forming frequency distributions of
clonotypes, identifying related subsets of clonotypes, displaying
clonotype frequency information, and the like.
[0064] After amplification of DNA from the genome (or amplification
of nucleic acid in the form of cDNA by reverse transcribing RNA),
the individual nucleic acid molecules can be isolated, optionally
re-amplified, and then sequenced individually. Exemplary
amplification protocols may be found in van Dongen et al. Leukemia,
17: 2257-2317 (2003) or van Dongen et al, U.S. patent publication
2006/0234234, which is incorporated by reference. Briefly, an
exemplary protocol is as follows: Reaction buffer: ABI Buffer II or
ABI Gold Buffer (Life Technologies, San Diego, Calif.): 50 .mu.L
final reaction volume; 100 ng sample DNA; 10 pmol of each primer
(subject to adjustments to balance amplification as described
below); dNTPs at 200 .mu.M final concentration; MgCl.sub.2 at 1.5
mM final concentration (subject to optimization depending on target
sequences and polymerase); Taq polymerase (1-2 U/tube): cycling
conditions: preactivation 7 min at 95.degree. C.; annealing at
60.degree. C.; cycling times: 30 s denaturation; 30 s annealing; 30
s extension. Polymerases that can be used for amplification in the
methods of the invention are commercially available and include,
for example, Taq polymerase, AccuPrime polymerase, or Pfu. The
choice of polymerase to use can be based on whether fidelity or
efficiency is preferred.
[0065] Real time PCR, picogreen staining, nanofluidic
electrophoresis (e.g. LabChip) or UV absorption measurements can be
used in an initial step to judge the functional amount of
amplifiable material.
[0066] In one aspect, multiplex amplifications are carried out so
that relative amounts of sequences in a starting population are
substantially the same as those in the amplified population, or
amplicon. That is, multiplex amplifications are carried out with
minimal amplification bias among member sequences of a sample
population. In one embodiment, such relative amounts are
substantially the same if each relative amount in an amplicon is
within five fold of its value in the starting sample. In another
embodiment, such relative amounts are substantially the same if
each relative amount in an amplicon is within two fold of its value
in the starting sample. As discussed more fully below,
amplification bias in PCR may be detected and corrected using
conventional techniques so that a set of PCR primers may be
selected for a predetermined repertoire that provide unbiased
amplification of any sample.
[0067] In regard to many repertoires based on TCR or BCR sequences,
a multiplex amplification optionally uses all the V segments. The
reaction is optimized to attempt to get amplification that
maintains the relative abundance of the sequences amplified by
different V segment primers. Some of the primers are related, and
hence many of the primers may "cross talk," amplifying templates
that are not perfectly matched with it. The conditions are
optimized so that each template can be amplified in a similar
fashion irrespective of which primer amplified it. In other words
if there are two templates, then after 1,000 fold amplification
both templates can be amplified approximately 1,000 fold, and it
does not matter that for one of the templates half of the amplified
products carried a different primer because of the cross talk. In
subsequent analysis of the sequencing data the primer sequence is
eliminated from the analysis, and hence it does not matter what
primer is used in the amplification as long as the templates are
amplified equally.
[0068] In one embodiment, amplification bias may be avoided by
carrying out a two-stage amplification (as described in Faham and
Willis, cited above) wherein a small number of amplification cycles
are implemented in a first, or primary, stage using primers having
tails non-complementary with the target sequences. The tails
include primer binding sites that are added to the ends of the
sequences of the primary amplicon so that such sites are used in a
second stage amplification using only a single forward primer and a
single reverse primer, thereby eliminating a primary cause of
amplification bias. Preferably, the primary PCR will have a small
enough number of cycles (e.g. 5-10) to minimize the differential
amplification by the different primers. The secondary amplification
is done with one pair of primers and hence the issue of
differential amplification is minimal. One percent of the primary
PCR is taken directly to the secondary PCR. Thirty-live cycles
(equivalent to .about.28 cycles without the 100 fold dilution step)
used between the two amplifications were sufficient to show a
robust amplification irrespective of whether the breakdown of
cycles were: one cycle primary and 34 secondary or 25 primary and
10 secondary. Even though ideally doing only 1 cycle in the primary
PCR may decrease the amplification bias, there are other
considerations. One aspect of this is representation. This plays a
role when the starting input amount is not in excess to the number
of reads ultimately obtained. For example, if 1,000,000 reads are
obtained and starting with 1,000,000 input molecules then taking
only representation from 100,000 molecules to the secondary
amplification would degrade the precision of estimating the
relative abundance of the different species in the original sample.
The 100 fold dilution between the 2 steps means that the
representation is reduced unless the primary PCR amplification
generated significantly more than 100 molecules. This indicates
that a minimum 8 cycles (256 fold), but more comfortably 10 cycle
(.about.1,000 fold), may be used. The alternative to that is to
take more than 1% of the primary PCR into the secondary but because
of the high concentration of primer used in the primary PCR, a big
dilution factor is can be used to ensure these primers do not
interfere in the amplification and worsen the amplification bias
between sequences. Another alternative is to add a purification or
enzymatic step to eliminate the primers from the primary PCR to
allow a smaller dilution of it. In this example, the primary PCR
was 10 cycles and the second 25 cycles.
Sequencing Populations of Recombined Nucleic Acids
[0069] Any high-throughput technique for sequencing nucleic acids
can be used in the method of the invention. Preferably, such
technique has a capability of generating in a cost-effective manner
a volume of sequence data from which at least 1000 clonotypes can
be determined, and preferably, from which at least 10,000 to
1,000,000 clonotypes can be determined. DNA sequencing techniques
include classic dideoxy sequencing reactions (Sanger method) using
labeled terminators or primers and gel separation in slab or
capillary, sequencing by synthesis using reversibly terminated
labeled nucleotides, pyrosequencing, 454 sequencing, allele
specific hybridization to a library of labeled oligonucleotide
probes, sequencing by synthesis using allele specific hybridization
to a library of labeled clones that is followed by ligation, real
time monitoring of the incorporation of labeled nucleotides during
a polymerization step, polony sequencing, and SOLiD sequencing.
Sequencing of the separated molecules has more recently been
demonstrated by sequential or single extension reactions using
polymerases or ligases as well as by single or sequential
differential hybridizations with libraries of probes. These
reactions have been performed on many clonal sequences in parallel
including demonstrations in current commercial applications of over
100 million sequences in parallel. These sequencing approaches can
thus be used to study the repertoire of T-cell receptor (TCR)
and/or B-cell receptor (BCR). In one aspect of the invention,
high-throughput methods of sequencing are employed that comprise a
step of spatially isolating individual molecules on a solid surface
where they are sequenced in parallel. Such solid surfaces may
include nonporous surfaces (such as in Solexa sequencing, e.g.
Bentley et al, Nature, 456: 53-59 (2008) or Complete Genomics
sequencing, e.g. Drmanac et al, Science, 327: 78-81 (2010)), arrays
of wells, which may include bead- or particle-bound templates (such
as with 454, e.g. Margulies et al, Nature, 437: 376-380 (2005) or
Ion Torrent sequencing, U.S. patent publication 2010/0137143 or
2010/0304982), micromachined membranes (such as with SMRT
sequencing, e.g. Eid et al, Science, 323: 133-138 (2009)), or bead
arrays (as with SOLiD sequencing or polony sequencing, e.g. Kim et
al. Science, 316: 1481-1414 (2007)). In another aspect, such
methods comprise amplifying the isolated molecules either before or
after they are spatially isolated on a solid surface. Prior
amplification may comprise emulsion-based amplification, such as
emulsion PCR, or rolling circle amplification. Of particular
interest is Solexa-based sequencing where individual template
molecules are spatially isolated on a solid surface, after which
they are amplified in parallel by bridge PCR to form separate
clonal populations, or clusters, and then sequenced, as described
in Bentley et al (cited above) and in manufacturer's instructions
(e.g. TruSeq.TM. Sample Preparation Kit and Data Sheet, Illumina,
Inc., San Diego, Calif., 2010); and further in the following
references: U.S. Pat. Nos. 6,090,592; 6,300,070; 7,115,400; and
EP0972081B1; which are incorporated by reference. In one
embodiment, individual molecules disposed and amplified on a solid
surface form clusters in a density of at least 10.sup.5 clusters
per cm.sup.2; or in a density of at least 5.times.10.sup.5 per
cm.sup.2; or in a density of at least 10.sup.6 clusters per
cm.sup.2. In one embodiment, sequencing chemistries are employed
having relatively high error rates. In such embodiments, the
average quality scores produced by such chemistries are
monotonically declining functions of sequence read lengths. In one
embodiment, such decline corresponds to 0.5 percent of sequence
reads have at least one error in positions 1-75; 1 percent of
sequence reads have at least one error in positions 76-100; and 2
percent of sequence reads have at least one error in positions
101-125.
[0070] In one aspect, a sequence-based clonotype profile of an
individual is obtained using the following steps: (a) obtaining a
nucleic acid sample from T-cells and/or B-cells of the individual;
(b) spatially isolating individual molecules derived from such
nucleic acid sample, the individual molecules comprising at least
one template generated from a nucleic acid in the sample, which
template comprises a somatically rearranged region or a portion
thereof, each individual molecule being capable of producing at
least one sequence read; (c) sequencing said spatially isolated
individual molecules; and (d) determining abundances of different
sequences of the nucleic acid molecules from the nucleic acid
sample to generate the clonotype profile. In some embodiments, the
method further includes one or more steps of amplifying the
individual molecules comprising recombined nucleic acids of T-cell
receptor genes or immunoglobulin genes. For example, such one or
more amplification steps may include a multi-stage PCR. In another
embodiment, the step of sequencing comprises bidirectionally
sequencing each of the spatially isolated individual molecules to
produce at least one forward sequence read and at least one reverse
sequence read. In another embodiment, the above method comprises
the following steps: (a) obtaining a nucleic acid sample from
T-cells and/or B-cells of the individual; (b) spatially isolating
individual molecules derived from such nucleic acid sample, the
individual molecules comprising nested sets of templates each
generated from a nucleic acid in the sample and each containing a
somatically rearranged region or a portion thereof, each nested set
being capable of producing a plurality of sequence reads each
extending in the same direction and each starting from a different
position on the nucleic acid from which the nested set was
generated; (c) sequencing said spatially isolated individual
molecules; and (d) determining abundances of different sequences of
the nucleic acid molecules from the nucleic acid sample to generate
the clonotype profile. In one embodiment, the step of sequencing
includes producing a plurality of sequence reads for each of the
nested sets. In another embodiment, each of the somatically
rearranged regions comprise a V region and a J region, and each of
the plurality of sequence reads starts from a different position in
the V region and extends in the direction of its associated J
region.
[0071] In one aspect, for each sample from an individual, the
sequencing technique used in the methods of the invention generates
sequences of least 1000 clonotypes per run; in another aspect, such
technique generates sequences of at least 10,000 clonotypes per
run; in another aspect, such technique generates sequences of at
least 100,000 clonotypes per run; in another aspect, such technique
generates sequences of at least 500,000 clonotypes per run; and in
another aspect, such technique generates sequences of at least
1,000,000 clonotypes per run. In still another aspect, such
technique generates sequences of between 100,000 to 1,000,000
clonotypes per run per individual sample.
[0072] The sequencing technique used in the methods of the provided
invention can generate about 30 bp, about 40 bp, about 50 bp, about
60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about
110, about 120 bp per read, about 150 bp, about 200 bp, about 250
bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, about
500 bp, about 550 bp, or about 600 bp per read.
[0073] While the present invention has been described with
reference to several particular example embodiments, those skilled
in the art will recognize that many changes may be made thereto
without departing from the spirit and scope of the present
invention. The present invention is applicable to a variety of
sensor implementations and other subject matter, in addition to
those discussed above.
DEFINITIONS
[0074] Unless otherwise specifically defined herein, terms and
symbols of nucleic acid chemistry, biochemistry, genetics, and
molecular biology used herein follow those of standard treatises
and texts in the field, e.g. Kornberg and Baker, DNA Replication.
Second Edition (W.H. Freeman, New York, 1992); Lehninger.
Biochemistry. Second Edition (Worth Publishers, New York, 1975):
Strachan and Read. Human Molecular Genetics, Second Edition
(Wiley-Liss, New York, 1999); Abbas et al, Cellular and Molecular
Immunology, 6.sup.th edition (Saunders, 2007).
[0075] "Aligning" in reference to two or more sequences, such as
sequence reads, means a method of comparing the two or more
sequences to determine how similar they are based on some sequence
distance measure. An exemplary method of aligning nucleotide
sequences is the Smith Waterman algorithm. Distance measures may
include Hamming distance. Levenshtein distance, or the like.
Distance measures may include a component related to the quality
values of nucleotides of the sequences being compared.
[0076] "Amplicon" means the product of a polynucleotide
amplification reaction; that is, a clonal population of
polynucleotides, which may be single stranded or double stranded,
which are replicated from one or more starting sequences. The one
or more starting sequences may be one or more copies of the same
sequence, or they may be a mixture of different sequences.
Preferably, amplicons are formed by the amplification of a single
starting sequence. Amplicons may be produced by a variety of
amplification reactions whose products comprise replicates of the
one or more starting, or target, nucleic acids. In one aspect,
amplification reactions producing amplicons are "template-driven"
in that base pairing of reactants, either nucleotides or
oligonucleotides, have complements in a template polynucleotide
that are required for the creation of reaction products. In one
aspect, template-driven reactions are primer extensions with a
nucleic acid polymerase or oligonucleotide ligations with a nucleic
acid ligase. Such reactions include, but are not limited to,
polymerase chain reactions (PCRs), linear polymerase reactions,
nucleic acid sequence-based amplification (NASBAs), rolling circle
amplifications, and the like, disclosed in the following references
that are incorporated herein by reference: Mullis et al, U.S. Pat.
Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et
al, U.S. Pat. No. 5,210,015 (real-time PCR with "taqman" probes);
Wittwer et al, U.S. Pat. No. 6,174,670; Kacian et al. U.S. Pat. No.
5,399,491 ("NASBA"); Lizardi, U.S. Pat. No. 5,854,033; Aono et al,
Japanese patent publ. JP 4-262799 (rolling circle amplification);
and the like. In one aspect, amplicons of the invention are
produced by PCRs. An amplification reaction may be a "real-time"
amplification if a detection chemistry is available that permits a
reaction product to be measured as the amplification reaction
progresses, e.g. "real-time PCR" described below, or "real-time
NASBA" as described in Leone et al, Nucleic Acids Research, 26:
2150-2155 (1998), and like references. As used herein, the term
"amplifying" means performing an amplification reaction. A
"reaction mixture" means a solution containing all the necessary
reactants for performing a reaction, which may include, but not be
limited to, buffering agents to maintain pH at a selected level
during a reaction, salts, co-factors, scavengers, and the like.
[0077] "Clonality" as used herein means a measure of the degree to
which the distribution of clonotype abundances among clonotypes of
a repertoire is skewed to a single or a few clonotypes. Roughly,
clonality is an inverse measure of clonotype diversity. Many
measures or statistics are available from ecology describing
species-abundance relationships that may be used for clonality
measures in accordance with the invention, e.g. Chapters 17 &
18, in Pielou, An Introduction to Mathematical Ecology.
(Wiley-Interscience, 1969). In one aspect, a clonality measure used
with the invention is a function of a clonotype profile (that is,
the number of distinct clonotypes detected and their abundances),
so that after a clonotype profile is measured, clonality may be
computed from it to give a single number. One clonality measure is
Simpson's measure, which is simply the probability that two
randomly drawn clonotypes will be the same. Other clonality
measures include information-based measures and McIntosh's
diversity index, disclosed in Pielou (cited above).
[0078] "Clonotype" means a recombined nucleotide sequence of a
lymphocyte which encodes an immune receptor or a portion thereof.
More particularly, clonotype means a recombined nucleotide sequence
of a T cell or B cell which encodes a T cell receptor (TCR) or B
cell receptor (BCR), or a portion thereof. In various embodiments,
clonotypes may encode all or a portion of a VDJ rearrangement of
IgH, a DJ rearrangement of IgH, a VJ rearrangement of IgK, a VJ
rearrangement of IgL, a VDJ rearrangement of TCR .beta., a DJ
rearrangement of TCR .beta., a VJ rearrangement of TCR .alpha., a
VJ rearrangement of TCR .gamma., a VDJ rearrangement of TCR
.delta., a VD rearrangement of TCR .delta., a Kde-V rearrangement,
or the like. Clonotypes may also encode translocation breakpoint
regions involving immune receptor genes, such as Bcl1-IgH or
Bcl1-IgH. In one aspect, clonotypes have sequences that are
sufficiently long to represent or reflect the diversity of the
immune molecules that they are derived from; consequently,
clonotypes may vary widely in length. In some embodiments,
clonotypes have lengths in the range of from 25 to 400 nucleotides;
in other embodiments, clonotypes have lengths in the range of from
25 to 200 nucleotides.
[0079] "Clonotype profile" means a listing of distinct clonotypes
and their relative abundances that are derived from a population of
lymphocytes. Typically, the population of lymphocytes are obtained
from a tissue sample. The term "clonotype profile" is related to,
but more general than, the immunology concept of immune
"repertoire" as described in references, such as the following:
Arstila et al. Science, 286: 958-961 (1999); Yassai et al.
Immunogenetics, 61: 493-502 (2009); Kedzierska et al, Mol. Immunol.
45(3): 607-618 (2008); and the like. The term "clonotype profile"
includes a wide variety of lists and abundances of rearranged
immune receptor-encoding nucleic acids, which may be derived from
selected subsets of lymphocytes (e.g. tissue-infiltrating
lymphocytes, immunophenotypic subsets, or the like), or which may
encode portions of immune receptors that have reduced diversity as
compared to full immune receptors. In some embodiments, clonotype
profiles may comprise at least 10.sup.3 distinct clonotypes; in
other embodiments, clonotype profiles may comprise at least
10.sup.4 distinct clonotypes; in other embodiments, clonotype
profiles may comprise at least 10.sup.5 distinct clonotypes; in
other embodiments, clonotype profiles may comprise at least
10.sup.6 distinct clonotypes. In such embodiments, such clonotype
profiles may further comprise abundances or relative frequencies of
each of the distinct clonotypes. In one aspect, a clonotype profile
is a set of distinct recombined nucleotide sequences (with their
abundances) that encode T cell receptors (TCRs) or B cell receptors
(BCRs), or fragments thereof, respectively, in a population of
lymphocytes of an individual, wherein the nucleotide sequences of
the set have a one-to-one correspondence with distinct lymphocytes
or their clonal subpopulations for substantially all of the
lymphocytes of the population. In one aspect, nucleic acid segments
defining clonotypes are selected so that their diversity (i.e. the
number of distinct nucleic acid sequences in the set) is large
enough so that substantially every T cell or B cell or clone
thereof in an individual carries a unique nucleic acid sequence of
such repertoire. That is, preferably each different clone of a
sample has different clonotype. In other aspects of the invention,
the population of lymphocytes corresponding to a repertoire may be
circulating B cells, or may be circulating T cells, or may be
subpopulations of either of the foregoing populations, including
but not limited to, CD4+ T cells, or CD8+ T cells, or other
subpopulations defined by cell surface markers, or the like. Such
subpopulations may be acquired by taking samples from particular
tissues, e.g. bone marrow, or lymph nodes, or the like, or by
sorting or enriching cells from a sample (such as peripheral blood)
based on one or more cell surface markers, size, morphology, or the
like. In still other aspects, the population of lymphocytes
corresponding to a repertoire may be derived from disease tissues,
such as a tumor tissue, an infected tissue, or the like. In one
embodiment, a clonotype profile comprising human TCR .beta. chains
or fragments thereof comprises a number of distinct nucleotide
sequences in the range of from 0.1.times.10.sup.6 to
1.8.times.10.sup.6, or in the range of from 0.5.times.10.sup.6 to
1.5.times.10.sup.6, or in the range of from 0.8.times.10.sup.6 to
1.2.times.10.sup.6. In another embodiment, a clonotype profile
comprising human IgH chains or fragments thereof comprises a number
of distinct nucleotide sequences in the range of from
0.1.times.10.sup.6 to 1.8.times.10.sup.6, or in the range of from
0.5.times.10.sup.6 to 1.5.times.10.sup.6, or in the range of from
0.8.times.10.sup.6 to 1.2.times.10.sup.6. In a particular
embodiment, a clonotype profile of the invention comprises a set of
nucleotide sequences encoding substantially all segments of the
V(D)J region of an IgH chain. In one aspect. "substantially all" as
used herein means every segment having a relative abundance of
0.001 percent or higher; or in another aspect. "substantially all"
as used herein means every segment having a relative abundance of
0.0001 percent or higher. In another particular embodiment, a
clonotype profile of the invention comprises a set of nucleotide
sequences that encodes substantially all segments of the V(D)J
region of a TCR .beta. chain. In another embodiment, a clonotype
profile of the invention comprises a set of nucleotide sequences
having lengths in the range of from 25-200 nucleotides and
including segments of the V, D, and J regions of a TCR .beta.
chain. In another embodiment, a clonotype profile of the invention
comprises a set of nucleotide sequences having lengths in the range
of from 25-200 nucleotides and including segments of the V, D, and
J regions of an IgH chain. In another embodiment, a clonotype
profile of the invention comprises a number of distinct nucleotide
sequences that is substantially equivalent to the number of
lymphocytes expressing a distinct IgH chain. In another embodiment,
a clonotype profile of the invention comprises a number of distinct
nucleotide sequences that is substantially equivalent to the number
of lymphocytes expressing a distinct TCR .beta. chain. In still
another embodiment, "substantially equivalent" means that with
ninety-nine percent probability a clonotype profile will include a
nucleotide sequence encoding an IgH or TCR .beta. or portion
thereof carried or expressed by every lymphocyte of a population of
an individual at a frequency of 0.001 percent or greater. In still
another embodiment. "substantially equivalent" means that with
ninety-nine percent probability a repertoire of nucleotide
sequences will include a nucleotide sequence encoding an IgH or TCR
.beta. or portion thereof carried or expressed by every lymphocyte
present at a frequency of 0.0001 percent or greater. In some
embodiments, clonotype profiles are derived from samples comprising
from 10.sup.5 to 10.sup.7 lymphocytes. Such numbers of lymphocytes
may be obtained from peripheral blood samples of from 1-10 mL.
[0080] "Coalescing" means treating two candidate clonotypes with
sequence differences as the same by determining that such
differences are due to experimental or measurement error and not
due to genuine biological differences. In one aspect, a sequence of
a higher frequency candidate clonotype is compared to that of a
lower frequency candidate clonotype and if predetermined criteria
are satisfied then the number of lower frequency candidate
clonotypes is added to that of the higher frequency candidate
clonotype and the lower frequency candidate clonotype is thereafter
disregarded. That is, the read counts associated with the lower
frequency candidate clonotype are added to those of the higher
frequency candidate clonotype.
[0081] "Complementarity determining regions" (CDRs) mean regions of
an immunoglobulin (i.e., antibody) or T cell receptor where the
molecule complements an antigen's conformation, thereby determining
the molecule's specificity and contact with a specific antigen. T
cell receptors and immunoglobulins each have three CDRs: CDR1 and
CDR2 are found in the variable (V) domain, and CDR3 includes some
of V, all of diverse (D) (heavy chains only) and joint (J), and
some of the constant (C) domains.
[0082] "Contamination" as used herein means the presence in a
sample taken from one individual at a given time of nucleic acid
from another individual or of nucleic acid from the same individual
from a sample taken at another time. In one aspect, "contamination"
means the presence of nucleic acid not originating from a given
patient tissue sample which may affect the interpretation of a
clonotype profile of the patient.
[0083] "Internal standard" means a nucleic acid sequence that is
processed in the same reaction as one or more target
polynucleotides in order to permit absolute or relative
quantification of the target polynucleotides in a sample. In one
aspect the reaction is an amplification reaction, such as PCR. An
internal standard may be endogenous or exogenous. That is, an
internal standard may occur naturally in the sample, or it may be
added to the sample prior to a reaction. In one aspect, one or more
exogenous internal standard sequences may be added to a reaction
mixture in predetermined concentrations to provide a calibration to
which an amplified sequence may be compared to determine the
quantity of its corresponding target polynucleotide in a sample.
Selection of the number, sequences, lengths, and other
characteristics of exogenous internal standards is a routine design
choice for one of ordinary skill in the art. Endogenous internal
standards, also referred to herein as "reference sequences." are
sequences natural to a sample that correspond to minimally
regulated genes that exhibit a constant and cell cycle-independent
level of transcription, e.g. Selvey et al, Mol. Cell Probes, 15:
307-311 (2001). Exemplary internal standards include, but are not
limited to, sequences from the following genes: GAPDH,
.rho.-microglobulin, 18S ribosomal RNA, and .beta.-actin.
[0084] "Kit" refers to any delivery system for delivering materials
or reagents for carrying out a method of the invention. In the
context of methods of the invention, such delivery systems include
systems that allow for the storage, transport, or delivery of
reaction reagents (e.g., primers, enzymes, internal standards, etc.
in the appropriate containers) and/or supporting materials (e.g.,
buffers, written instructions for performing the assay etc.) from
one location to another. For example, kits include one or more
enclosures (e.g., boxes) containing the relevant reaction reagents
and/or supporting materials. Such contents may be delivered to the
intended recipient together or separately. For example, a first
container may contain an example for use in an assay, while a
second container contains primers.
[0085] "Lymphoid neoplasm" means an abnormal proliferation of
lymphocytes that may be malignant or non-malignant. A lymphoid
cancer is a malignant lymphoid neoplasm. Lymphoid neoplasms are the
result of, or are associated with, lymphoproliferative disorders,
including but not limited to, follicular lymphoma, chronic
lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), hairy
cell leukemia, lymphomas, multiple myeloma, post-transplant
lymphoproliferative disorder, mantle cell lymphoma (MCL), diffuse
large B cell lymphoma (DLBCL). T cell lymphoma, or the like, e.g.
Jaffe et al, Blood, 112: 4384-4399 (2008): Swerdlow et al, WHO
Classification of Tumours of Haematopoietic and Lymphoid Tissues
(e. 4.sup.th) (IARC Press, 2008).
[0086] "Minimal residual disease" means remaining cancer cells
after treatment. The term is most frequently used in connection
with treatment of lymphomas and leukemias.
[0087] "Percent homologous," "percent identical," or like terms
used in reference to the comparison of a reference sequence and
another sequence ("comparison sequence") mean that in an optimal
alignment between the two sequences, the comparison sequence is
identical to the reference sequence in a number of subunit
positions equivalent to the indicated percentage, the subunits
being nucleotides for polynucleotide comparisons or amino acids for
polypeptide comparisons. As used herein, an "optimal alignment" of
sequences being compared is one that maximizes matches between
subunits and minimizes the number of gaps employed in constructing
an alignment. Percent identities may be determined with
commercially available implementations of algorithms, such as that
described by Needleman and Wunsch, J. Mol. Biol., 48: 443-453
(1970)("GAP" program of Wisconsin Sequence Analysis Package.
Genetics Computer Group, Madison, Wis.), or the like. Other
software packages in the art for constructing alignments and
calculating percentage identity or other measures of similarity
include the "BestFit" program, based on the algorithm of Smith and
Waterman, Advances in Applied Mathematics, 2: 482-489 (1981)
(Wisconsin Sequence Analysis Package, Genetics Computer Group,
Madison, Wis.). In other words, for example, to obtain a
polynucleotide having a nucleotide sequence at least 95 percent
identical to a reference nucleotide sequence, up to five percent of
the nucleotides in the reference sequence may be deleted or
substituted with another nucleotide, or a number of nucleotides up
to five percent of the total number of nucleotides in the reference
sequence may be inserted into the reference sequence.
[0088] "Polymerase chain reaction," or "PCR," means a reaction for
the in vitro amplification of specific DNA sequences by the
simultaneous primer extension of complementary strands of DNA. In
other words, PCR is a reaction for making multiple copies or
replicates of a target nucleic acid flanked by primer binding
sites, such reaction comprising one or more repetitions of the
following steps: (i) denaturing the target nucleic acid, (ii)
annealing primers to the primer binding sites, and (iii) extending
the primers by a nucleic acid polymerase in the presence of
nucleoside triphosphates. Usually, the reaction is cycled through
different temperatures optimized for each step in a thermal cycler
instrument. Particular temperatures, durations at each step, and
rates of change between steps depend on many factors well-known to
those of ordinary skill in the art, e.g. exemplified by the
references: McPherson et al, editors. PCR: A Practical Approach and
PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995,
respectively). For example, in a conventional PCR using Taq DNA
polymerase, a double stranded target nucleic acid may be denatured
at a temperature >90.degree. C., primers annealed at a
temperature in the range 50-75.degree. C., and primers extended at
a temperature in the range 72-78.degree. C. The term "PCR"
encompasses derivative forms of the reaction, including but not
limited to, RT-PCR, real-lime PCR, nested PCR, quantitative PCR,
multiplexed PCR, and the like. The particular format of PCR being
employed is discernible by one skilled in the art from the context
of an application. Reaction volumes range from a few hundred
nanoliters, e.g. 200 nL, to a few hundred .mu.L, e.g. 200 .mu.L.
"Reverse transcription PCR," or "RT-PCR," means a PCR that is
preceded by a reverse transcription reaction that converts a target
RNA to a complementary single stranded DNA, which is then
amplified, e.g. Tecott et al, U.S. Pat. No. 5,168,038, which patent
is incorporated herein by reference. "Real-time PCR" means a PCR
for which the amount of reaction product, i.e. amplicon, is
monitored as the reaction proceeds. There are many forms of
real-time PCR that differ mainly in the detection chemistries used
for monitoring the reaction product, e.g. Gelfand et al, U.S. Pat.
No. 5,210,015 ("taqman"); Wittwer et al, U.S. Pat. Nos. 6,174,670
and 6,569,627 (intercalating dyes): Tyagi et al. U.S. Pat. No.
5,925,517 (molecular beacons); which patents are incorporated
herein by reference. Detection chemistries for real-time PCR are
reviewed in Mackay et al, Nucleic Acids Research, 30: 1292-1305
(2002), which is also incorporated herein by reference. "Nested
PCR" means a two-stage PCR wherein the amplicon of a first PCR
becomes the sample for a second PCR using a new set of primers, at
least one of which binds to an interior location of the first
amplicon. As used herein, "initial primers" in reference to a
nested amplification reaction mean the primers used to generate a
first amplicon, and "secondary primers" mean the one or more
primers used to generate a second, or nested, amplicon.
"Multiplexed PCR" means a PCR wherein multiple target sequences (or
a single target sequence and one or more reference sequences) are
simultaneously carried out in the same reaction mixture, e.g.
Bernard et al, Anal. Biochem., 273: 221-228 (1999) (two-color
real-time PCR). Usually, distinct sets of primers are employed for
each sequence being amplified. Typically, the number of target
sequences in a multiplex PCR is in the range of from 2 to 50, or
from 2 to 40, or from 2 to 30. "Quantitative PCR" means a PCR
designed to measure the abundance of one or more specific target
sequences in a sample or specimen. Quantitative PCR includes both
absolute quantitation and relative quantitation of such target
sequences. Quantitative measurements are made using one or more
reference sequences or internal standards that may be assayed
separately or together with a target sequence. The reference
sequence may be endogenous or exogenous to a sample or specimen,
and in the latter case, may comprise one or more competitor
templates. Typical endogenous reference sequences include segments
of transcripts of the following genes: .beta.-actin. GAPDH,
.beta..sub.2-microglobulin, ribosomal RNA, and the like. Techniques
for quantitative PCR are well-known to those of ordinary skill in
the art, as exemplified in the following references that are
incorporated by reference: Freeman et al, Biotechniques, 26:
112-126 (1999): Becker-Andre et al, Nucleic Acids Research, 17:
9437-9447 (1989); Zimmerman et al, Biotechniques, 21: 268-279
(1996): Diviacco et al, Gene, 122: 3013-3020 (1992); Becker-Andre
et al, Nucleic Acids Research, 17: 9437-9446 (1989); and the
like.
[0089] "Polynucleotide" refers to a linear polymer of nucleotide
monomers and may be DNA or RNA. Monomers making up polynucleotides
are capable of specifically binding to a natural polynucleotide by
way of a regular pattern of monomer-to-monomer interactions, such
as Watson-Crick type of base pairing, base stacking, Hoogsteen or
reverse Hoogsteen types of base pairing, or the like. Such monomers
and their internucleosidic linkages may be naturally occurring or
may be analogs thereof, e.g., naturally occurring or non-naturally
occurring analogs. Non-naturally occurring analogs may include
PNAs, phosphorothioate internucleosidic linkages, bases containing
linking groups permitting the attachment of labels, such as
fluorophores, or haptens, and the like. Polynucleotides may
comprise the four natural nucleosides (e.g., deoxyadenosine,
deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their
ribose counterparts for RNA) linked by phosphodiester linkages.
However, they may also comprise non-natural nucleotide analogs,
e.g. including modified bases, sugars, or internucleosidic
linkages. It is clear to those skilled in the art that where an
enzyme has specific oligonucleotide or polynucleotide substrate
requirements for activity (e.g. single stranded DNA. RNA/DNA
duplex, or the like), then selection of an appropriate composition
for the oligonucleotide or polynucleotide substrates is well within
the knowledge of one of ordinary skill, especially with guidance
from treatises such as Sambrook et al, MOLECULAR CLONING, 2nd ed.
(Cold Spring Harbor Laboratory, New York, 1989), and like
references. As used herein, the term "oligonucleotide" refers to
smaller polynucleotides, for example, having 3-60 monomeric units,
or in some embodiments having from 12 to 60 monomeric units. In
various embodiments, a polynucleotide or oligonucleotides may be
represented by a sequence of letters (upper or lower case), such as
"ATGCCTG," and it will be understood that the nucleotides are in
5'.fwdarw.3' order from left to right and that "A" denotes
deoxyadenosine, "C" denotes deoxycytidine. "G" denotes
deoxyguanosine, and "T" denotes thymidine. "I" denotes
deoxyinosine. "U" denotes uridine, unless otherwise indicated or
obvious from context.
[0090] "Primer" means an oligonucleotide, either natural or
synthetic that is capable, upon forming a duplex with a
polynucleotide template, of acting as a point of initiation of
nucleic acid synthesis and being extended from its 3' end along the
template so that an extended duplex is formed. Extension of a
primer is usually carried out with a nucleic acid polymerase, such
as a DNA or RNA polymerase. The sequence of nucleotides added in
the extension process is determined by the sequence of the template
polynucleotide. Usually primers are extended by a DNA polymerase.
Primers usually have a length in the range of from 14 to 40
nucleotides, or in the range of from 18 to 36 nucleotides. Primers
are employed in a variety of nucleic amplification reactions, for
example, linear amplification reactions using a single primer, or
polymerase chain reactions, employing two or more primers. Guidance
for selecting the lengths and sequences of primers for particular
applications is well known to those of ordinary skill in the art,
as evidenced by the following references that are incorporated by
reference: Dieffenbach, editor, PCR Primer: A Laboratory Manual,
2.sup.nd Edition (Cold Spring Harbor Press, New York, 2003).
[0091] "Quality score" means a measure of the probability that a
base assignment at a particular sequence location is correct. A
variety methods are well known to those of ordinary skill for
calculating quality scores for particular circumstances, such as,
for bases called as a result of different sequencing chemistries,
detection systems, base-calling algorithms, and so on. Generally,
quality score values are monotonically related to probabilities of
correct base calling. For example, a quality score, or Q, of 10 may
mean that there is a 90 percent chance that a base is called
correctly, a Q of 20 may mean that there is a 99 percent chance
that a base is called correctly, and so on. For some sequencing
platforms, particularly those using sequencing-by-synthesis
chemistries, average quality scores decrease as a function of
sequence read length, so that quality scores at the beginning of a
sequence read are higher than those at the end of a sequence read,
such declines being due to phenomena such as incomplete extensions,
carry forward extensions, loss of template, loss of polymerase,
capping failures, deprotection failures, and the like.
[0092] "Sequence read" means a sequence of nucleotides determined
from a sequence or stream of data generated by a sequencing
technique, which determination is made, for example, by means of
base-calling software associated with the technique, e.g.
base-calling software from a commercial provider of a DNA
sequencing platform. A sequence read usually includes quality
scores for each nucleotide in the sequence. Typically, sequence
reads are made by extending a primer along a template nucleic acid,
e.g. with a DNA polymerase or a DNA ligase. Data is generated by
recording signals, such as optical, chemical (e.g. pH change), or
electrical signals, associated with such extension. Such initial
data is converted into a sequence read.
[0093] "Sequence tag" (or "tag") or "barcode" means an
oligonucleotide that is attached to a polynucleotide or template
molecule and is used to identify and/or track the polynucleotide or
template in a reaction or a series of reactions. A sequence tag may
be attached to the 3'- or 5'-end of a polynucleotide or template or
it may be inserted into the interior of such polynucleotide or
template to form a linear conjugate, sometime referred to herein as
a "tagged polynucleotide." or "tagged template." or
"tag-polynucleotide conjugate," "tag-molecule conjugate," or the
like. Sequence tags may vary widely in size and compositions; the
following references, which are incorporated herein by reference,
provide guidance for selecting sets of sequence tags appropriate
for particular embodiments: Brenner, U.S. Pat. No. 5,635,400;
Brenner and Macevicz, U.S. Pat. No. 7,537,897; Brenner et al, Proc.
Natl. Acad. Sci., 97: 1665-1670 (2000); Church et al. European
patent publication 0 303 459; Shoemaker et al, Nature Genetics, 14:
450-456 (1996); Morris et al, European patent publication
0799897A1; Wallace, U.S. Pat. No. 5,981,179: and the like. Lengths
and compositions of sequence tags can vary widely, and the
selection of particular lengths and/or compositions depends on
several factors including, without limitation, how tags are used to
generate a readout, e.g. via a hybridization reaction or via an
enzymatic reaction, such as sequencing; whether they are labeled,
e.g. with a fluorescent dye or the like; the number of
distinguishable oligonucleotide tags required to unambiguously
identify a set of polynucleotides, and the like, and how different
must tags of a set be in order to ensure reliable identification.
e.g. freedom from cross hybridization or misidentification from
sequencing errors. In one aspect, sequence tags can each have a
length within a range of from 2 to 36 nucleotides, or from 4 to 30
nucleotides, or from 8 to 20 nucleotides, or from 6 to 10
nucleotides, respectively. In one aspect, sets of sequence tags are
used wherein each sequence tag of a set has a unique nucleotide
sequence that differs from that of every other tag of the same set
by at least two bases; in another aspect, sets of sequence tags are
used wherein the sequence of each tag of a set differs from that of
every other tag of the same set by at least three bases.
[0094] "Sequence tree" means a tree data structure for representing
nucleotide sequences. In one aspect, a tree data structure of the
invention is a rooted directed tree comprising nodes and edges that
do not include cycles, or cyclical pathways. Edges from nodes of
tree data structures of the invention are usually ordered. Nodes
and/or edges are structures that may contain, or be associated
with, a value. Each node in a tree has zero or more child nodes,
which by convention are shown below it in the tree. A node that has
a child is called the child's parent node. A node has at most one
parent. Nodes that do not have any children are called leaf nodes.
The topmost node in a tree is called the root node. Being the
topmost node, the root node will not have parents. It is the node
at which operations on the tree commonly begin (although some
algorithms begin with the leaf nodes and work up ending at the
root). All other nodes can be reached from it by following edges or
links.
Sequence CWU 1
1
6124DNAArtificial Sequenceprimer 1agttctggct aacctgtaga gcca
24224DNAArtificial Sequenceprimer 2agttcgggct aacctgtcga gcca
24324DNAArtificial Sequenceprimer 3agttccggct aacctgtcga gcca
24422DNAArtificial Sequenceprimer 4nnnnnnnnnn nnnnnnnnnn nn
22512DNAArtificial Sequenceprimer 5gtattttttt ct 12613DNAArtificial
Sequenceprimer 6ttcagggggg gct 13
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