U.S. patent application number 17/371378 was filed with the patent office on 2021-11-04 for methods for detecting mutation load from a tumor sample.
The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Ruchi Chaudhary, Fiona HYLAND.
Application Number | 20210343367 17/371378 |
Document ID | / |
Family ID | 1000005712863 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210343367 |
Kind Code |
A1 |
Chaudhary; Ruchi ; et
al. |
November 4, 2021 |
METHODS FOR DETECTING MUTATION LOAD FROM A TUMOR SAMPLE
Abstract
A targeted panel with low sample input requirements from a tumor
only sample may be processed to estimate mutation load in a tumor
sample. The method may include detecting variants in nucleic acid
sequence reads corresponding to targeted locations in the tumor
sample genome; annotating detected variants with an annotation
information from a population database; filtering the detected
variants, wherein the filtering rule set retains the somatic
variants and removes germ-line variants; counting the identified
somatic variants to give a number of somatic variants; determining
a number of bases in covered regions of the targeted locations in
the tumor sample genome; and calculating a number of somatic
variants per megabase, provides an estimate of the mutation load
per megabase in the tumor sample genome.
Inventors: |
Chaudhary; Ruchi; (Redwood
City, CA) ; HYLAND; Fiona; (San Mateo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Family ID: |
1000005712863 |
Appl. No.: |
17/371378 |
Filed: |
July 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15834520 |
Dec 7, 2017 |
11101019 |
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17371378 |
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62585598 |
Nov 14, 2017 |
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62579645 |
Oct 31, 2017 |
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62431493 |
Dec 8, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G16B 20/40 20190201;
G16B 20/00 20190201; G16B 30/10 20190201; G16B 30/00 20190201; G16B
20/20 20190201 |
International
Class: |
G16B 20/20 20060101
G16B020/20; G16B 20/00 20060101 G16B020/00; G16B 30/00 20060101
G16B030/00; G16B 30/10 20060101 G16B030/10 |
Claims
1. A method of analyzing a tumor sample genome for a mutation load,
comprising: amplifying nucleic acid sequences at targeted locations
in the tumor sample genome by a targeted panel with a low sample
input from the tumor sample to generate a plurality of nucleic acid
sequence reads, wherein the tumor sample is a tumor only sample;
detecting variants in the plurality of nucleic acid sequence reads
to produce a plurality of detected variants; filtering the
plurality of detected variants, wherein the filtering applies a
rule set to the detected variants to retain somatic variants;
calculating a tumor mutation load in the tumor only sample by
dividing a number of the somatic variants by a number of bases in
covered regions of the targeted locations.
2. The method of claim 1, wherein the low sample input comprises 1
ng to 40 ng of sample DNA.
3. The method of claim 1, wherein the low sample input comprises 1
ng to 20 ng of sample DNA.
4. The method of claim 1, wherein the low sample input comprises 10
ng to 20 ng of sample DNA.
5. The method of claim 1, wherein the targeted panel covers
approximately 1.7 Mb of the tumor sample genome.
6. The method of claim 1, further comprising annotating one or more
detected variants of the plurality of detected variants with an
annotation information from one or more population databases,
wherein the population databases include information associated
with variants in a population.
7. The method of claim 6, wherein the annotation information
includes a minor allele frequency associated with a given
variant.
8. The method of claim 1, wherein the rule set includes retaining
the detected variants whose minor allele frequency (MAF) is within
a MAF range.
9. The method of claim 8, wherein the MAF range is from 0 to
10.sup.-6.
10. The method of claim 8, wherein the MAF range is from 0 to
0.01.
11. The method of claim 1, wherein the rule set further comprises
retaining the detected variants that are single nucleotide variants
(SNVs).
12. The method of claim 1, wherein the rule set further comprises
retaining the detected variants that are SNVs, insertion variants
and deletion variants (indels).
13. The method of claim 1, wherein the rule set further comprises
removing the detected variants that are SNVs corresponding to SNPs
in a UCSC Common SNP database.
14. The method of claim 1, wherein the rule set further comprises
removing the detected variants in regions having homopolymer
lengths greater than seven.
15. The method of claim 1, wherein the step of detecting variants
is configured by a variant caller parameter including a minimum
coverage parameter.
16. The method of claim 15, wherein the minimum coverage parameter
is in a range from 20 to 60.
17. The method of claim 1, wherein the step of detecting variants
is configured by a variant caller parameter including a minimum
allele frequency parameter.
18. The method of claim 1, wherein the step of detecting variants
is configured by a variant caller parameter including a strand bias
parameter.
19. The method of claim 1, wherein the step of detecting variants
is configured by a variant caller parameter including a data
quality stringency parameter.
20. The method of claim 1, further comprising associating sources
of somatic mutation damage with the somatic variants.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 15/834,520 filed Dec. 7, 2017, which claims priority to U.S.
application Nos. 62/585,598 filed Nov. 14, 2017, U.S. application
No. 62/579,645 filed Oct. 31, 2017, and U.S. application No.
62/431,493 filed Dec. 8, 2016, which disclosures are herein
incorporated by reference in their entirety.
BRIEF SUMMARY OF THE INVENTION
[0002] High tumor mutation load is a biomarker that shown in some
cancer types to predict positive response to immune checkpoint
inhibitors. Current methods to estimate tumor mutation load may
require large amounts of DNA to support whole exome sequencing and
matched tumor and normal samples. A targeted panel with low sample
input requirements from a tumor sample may be used to estimate
mutation load in a tumor sample genome.
[0003] According to an exemplary embodiment, there is provided a
method of analyzing a tumor sample genome for a mutation load,
including the following steps: (1) detecting variants in a
plurality of nucleic acid sequence reads to produce a plurality of
detected variants, wherein the nucleic acid sequence reads
correspond to a plurality of targeted locations in the tumor sample
genome, wherein the detected variants include somatic variants and
germ-line variants; (2) annotating one or more detected variants of
the plurality of detected variants with an annotation information
from one or more population databases, wherein the population
databases include information associated with variants in a
population, wherein the annotation information includes a minor
allele frequency associated with a given variant; (3) filtering the
plurality of detected variants, wherein the filtering applies a
rule set to the detected variants to retain the somatic variants,
the rule set including retaining the detected variants whose minor
allele frequency (MAF) is within a MAF range, wherein the filtering
produces identified somatic variants; (4) counting the identified
somatic variants to give a number of somatic variants; (5)
determining a number of bases in covered regions of the targeted
locations in the tumor sample genome; and (6) calculating a number
of somatic variants per megabase by dividing the number of
identified somatic variants by the number of bases in the covered
regions to produce the mutation load for the tumor sample
genome.
[0004] According to an exemplary embodiment, there is provided a
system for analyzing a tumor sample genome for a mutation load,
comprising a processor and a data store communicatively connected
with the processor, the processor configured to perform the steps
including: detecting variants in a plurality of nucleic acid
sequence reads to produce a plurality of detected variants, wherein
the nucleic acid sequence reads correspond to a plurality of
targeted locations in the tumor sample genome, wherein the detected
variants include somatic variants and germ-line variants;
annotating one or more detected variants of the plurality of
detected variants with an annotation information from one or more
population databases stored in the data store, wherein the
population databases include information associated with variants
in a population, wherein the annotation information includes a
minor allele frequency associated with a given variant; filtering
the plurality of detected variants, wherein the filtering applies a
rule set to the detected variants to retain the somatic variants,
the rule set including retaining the detected variants whose minor
allele frequency (MAF) is within a MAF range, wherein the filtering
produces identified somatic variants; counting the identified
somatic variants to give a number of somatic variants; determining
a number of bases in covered regions of the targeted locations in
the tumor sample genome; and calculating a number of somatic
variants per megabase by dividing the number of identified somatic
variants by the number of bases in the covered regions to produce
the mutation load for the tumor sample genome.
[0005] According to an exemplary embodiment, there is provided a
non-transitory machine-readable storage medium comprising
instructions which, when executed by a processor, cause the
processor to perform a method for analyzing a tumor sample genome
for a mutation load, including: (1) detecting variants in a
plurality of nucleic acid sequence reads to produce a plurality of
detected variants, wherein the nucleic acid sequence reads
correspond to a plurality of targeted locations in the tumor sample
genome, wherein the detected variants include somatic variants and
germ-line variants; (2) annotating one or more detected variants of
the plurality of detected variants with an annotation information
from one or more population databases, wherein the population
databases include information associated with variants in a
population, wherein the annotation information includes a minor
allele frequency associated with a given variant; (3) filtering the
plurality of detected variants, wherein the filtering applies a
rule set to the detected variants to retain the somatic variants,
the rule set including retaining the detected variants whose minor
allele frequency (MAF) is within a MAF range, wherein the filtering
produces identified somatic variants; (4) counting the identified
somatic variants to give a number of somatic variants; (5)
determining a number of bases in covered regions of the targeted
locations in the tumor sample genome; and (6) calculating a number
of somatic variants per megabase by dividing the number of
identified somatic variants by the number of bases in the covered
regions to produce the mutation load for the tumor sample
genome.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] 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 will be 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:
[0007] FIG. 1 is a block diagram of a method of detecting tumor
mutation load, according to an exemplary embodiment.
[0008] FIG. 2A is an example of a histogram plot of the number of
variants per allele ratio including all detected variants, both
germ-line and somatic variants.
[0009] FIG. 2B is an example of a histogram plot of the number of
variants per allele ratio after applying the filter rule set to
remove the germ-line variants.
[0010] FIG. 3A is an example of a histogram plot of the number of
variants per allele ratio including all detected variants, both
germ-line and somatic variants.
[0011] FIG. 3B is an example of a histogram plot of the number of
variants per allele ratio after applying the filter rule set to
remove the germ-line variants.
[0012] FIG. 4A is an example of a histogram plot of the number of
variants per allele ratio including all detected variants, both
germ-line and somatic variants.
[0013] FIG. 4B is an example of a histogram plot of the number of
variants per allele ratio after applying the filter rule set to
remove the germ-line variants.
[0014] FIG. 5 shows an example of comparisons of somatic SNV
mutation counts resulting from analysis of tumor sample only and
analysis of matched tumor-normal samples.
[0015] FIG. 6 shows an example of comparisons of somatic SNV
mutation counts resulting from analysis of tumor sample only and
analysis of matched tumor-normal samples.
[0016] FIG. 7 shows the results of testing the reproducibility of
the somatic mutation counts.
[0017] FIG. 8 shows a plot comparing the mutation count through the
targeted panel for TML versus the mutation count through WES.
[0018] FIG. 9A shows the comparison of mutation counts between the
targeted panel and WES for melanoma.
[0019] FIG. 9B shows the comparison of mutation counts between the
targeted panel and WES for lung cancer.
[0020] FIG. 9C shows the comparison of mutation counts between the
targeted panel and WES for colorectal cancer.
[0021] FIG. 9D shows the comparison of mutation counts between the
targeted panel and WES for endometrial cancer.
[0022] FIG. 10A shows an example of box plots for the WES TML
counts versus clinical response.
[0023] FIG. 10B shows an example of box plots for the somatic
mutation counts intersecting with the targeted panel versus
clinical response.
[0024] FIG. 11A shows an example of box plots for the WES TML
counts versus clinical response.
[0025] FIG. 11B shows an example of box plots for the somatic
mutation counts intersecting with the targeted panel versus
clinical response.
[0026] FIG. 12 shows an example of mutation counts per Mb for MSI
high and MSI low samples.
[0027] FIG. 13 is a pie chart for the pattern of somatic mutations
for the example of percentage values given in Table 4.
[0028] FIG. 14 is a schematic diagram of an exemplary system for
reconstructing a nucleic acid sequence, in accordance with various
embodiments.
[0029] FIG. 15 is a schematic diagram of a system for annotating
genomic variants, in accordance with various embodiments.
DETAILED DESCRIPTION OF THE INVENTION
[0030] In accordance with the teachings and principles embodied in
this application, new methods, systems and non-transitory
machine-readable storage medium are provided to estimate tumor
mutation load by analysis of nucleic acid sequence reads from a
tumor only sample genome.
[0031] In various embodiments, DNA (deoxyribonucleic acid) may be
referred to as a chain of nucleotides consisting of 4 types of
nucleotides; A (adenine), T (thymine), C (cytosine), and G
(guanine), and that RNA (ribonucleic acid) is comprised of 4 types
of nucleotides; A, U (uracil), G, and C. Certain pairs of
nucleotides specifically bind to one another in a complementary
fashion (called complementary base pairing). That is, adenine (A)
pairs with thymine (T) (in the case of RNA, however, adenine (A)
pairs with uracil (U)), and cytosine (C) pairs with guanine (G).
When a first nucleic acid strand binds to a second nucleic acid
strand made up of nucleotides that are complementary to those in
the first strand, the two strands bind to form a double strand. In
various embodiments, "nucleic acid sequencing data," "nucleic acid
sequencing information," "nucleic acid sequence," "genomic
sequence," "genetic sequence," or "fragment sequence," "nucleic
acid sequence read" or "nucleic acid sequencing read" denotes any
information or data that is indicative of the order of the
nucleotide bases (e.g., adenine, guanine, cytosine, and
thymine/uracil) in a molecule (e.g., whole genome, whole
transcriptome, exome, oligonucleotide, polynucleotide, fragment,
etc.) of DNA or RNA. It should be understood that the present
teachings contemplate sequence information obtained using all
available varieties of techniques, platforms or technologies,
including, but not limited to: capillary electrophoresis,
microarrays, ligation-based systems, polymerase-based systems,
hybridization-based systems, direct or indirect nucleotide
identification systems, pyrosequencing, ion- or pH-based detection
systems, electronic signature-based systems, etc.
[0032] The phrase "base space" refers to a nucleic acid sequence
data schema where nucleic acid sequence information is represented
by the actual nucleotide base composition of the nucleic acid
sequence. For example, the nucleic acid sequence "ATCGA" is
represented in base space by the actual nucleotide base identities
(for example, A, T/or U, C, G) of the nucleic acid sequence.
[0033] The phrase "flow space" refers to a nucleic acid sequence
data schema wherein nucleic acid sequence information is
represented by nucleotide base identifications (or identifications
of known nucleotide base flows) coupled with signal or numerical
quantification components representative of nucleotide
incorporation events for the nucleic acid sequence. The
quantification components may be related to the relative number of
continuous base repeats, such as homopolymers, whose incorporation
is associated with a respective nucleotide base flow. For example,
the nucleic acid sequence "ATTTGA" may be represented by the
nucleotide base identifications A, T, G and A (based on the
nucleotide base flow order) plus a quantification component for the
various flows indicating base presence/absence as well as possible
existence of homopolymers. Thus for "T" in the example sequence
above, the quantification component may correspond to a signal or
numerical identifier of greater magnitude than would be expected
for a single "T" and may be resolved to indicate the presence of a
homopolymer stretch of "T"s (in this case a 3-mer) in the "ATTTGA"
nucleic acid sequence.
[0034] A "polynucleotide", "nucleic acid", or "oligonucleotide"
refers to a linear polymer of nucleosides (including
deoxyribonucleosides, ribonucleosides, or analogs thereof) joined
by internucleosidic linkages. Typically, a polynucleotide comprises
at least three nucleosides. Usually oligonucleotides range in size
from a few monomeric units, for example 3-4, to several hundreds of
monomeric units. Whenever a polynucleotide such as an
oligonucleotide is represented by a sequence of letters, such as
"ATGCCTG," it will be understood that the nucleotides are in
5'->3' order from left to right and that "A" denotes
deoxyadenosine, "C" denotes deoxycytidine, "G" denotes
deoxyguanosine, and "T" denotes thymidine, unless otherwise noted.
The letters A, C, G, and T may be used to refer to the bases
themselves, to nucleosides, or to nucleotides comprising the bases,
as is standard in the art.
[0035] The phrase "genomic variants" or "genome variants" denote a
single or a grouping of sequences (in DNA or RNA) that have
undergone changes as referenced against a particular species or
sub-populations within a particular species due to mutations,
recombination/crossover or genetic drift. Examples of types of
genomic variants include, but are not limited to: single nucleotide
polymorphisms (SNPs), copy number variations (CNVs),
insertions/deletions (Indels), inversions, etc.
[0036] In various embodiments, genomic variants can be detected
using a nucleic acid sequencing system and/or analysis of
sequencing data. The sequencing workflow can begin with the test
sample being sheared or digested into hundreds, thousands or
millions of smaller fragments which are sequenced on a nucleic acid
sequencer to provide hundreds, thousands or millions of sequence
reads, such as nucleic acid sequence reads. Each read can then be
mapped to a reference or target genome, and in the case of
mate-pair fragments, the reads can be paired thereby allowing
interrogation of repetitive regions of the genome. The results of
mapping and pairing can be used as input for various standalone or
integrated genome variant (for example, SNP, CNV, Indel, inversion,
etc.) analysis tools.
[0037] The phrase "sample genome" can denote a whole or partial
genome of an organism.
[0038] The term "allele" as used herein refers to a genetic
variation associated with a gene or a segment of DNA, i.e., one of
two or more alternate forms of a DNA sequence occupying the same
locus.
[0039] The term "locus" as used herein refers to a specific
position on a chromosome or a nucleic acid molecule. Alleles of a
locus are located at identical sites on homologous chromosomes.
[0040] As used herein, a "targeted panel" refers to a set of
target-specific primers that are designed for selective
amplification of target gene sequences in a sample. In some
embodiments, following selective amplification of at least one
target sequence, the workflow further includes nucleic acid
sequencing of the amplified target sequence.
[0041] As used herein, "target sequence" or "target gene sequence"
and its derivatives, refers to any single or double-stranded
nucleic acid sequence that can be amplified or synthesized
according to the disclosure, including any nucleic acid sequence
suspected or expected to be present in a sample. In some
embodiments, the target sequence is present in double-stranded form
and includes at least a portion of the particular nucleotide
sequence to be amplified or synthesized, or its complement, prior
to the addition of target-specific primers or appended adapters.
Target sequences can include the nucleic acids to which primers
useful in the amplification or synthesis reaction can hybridize
prior to extension by a polymerase. In some embodiments, the term
refers to a nucleic acid sequence whose sequence identity, ordering
or location of nucleotides is determined by one or more of the
methods of the disclosure.
[0042] As used herein, "target-specific primer" and its
derivatives, refers to a single stranded or double-stranded
polynucleotide, typically an oligonucleotide, that includes at
least one sequence that is at least 50% complementary, typically at
least 75% complementary or at least 85% complementary, more
typically at least 90% complementary, more typically at least 95%
complementary, more typically at least 98% or at least 99%
complementary, or identical, to at least a portion of a nucleic
acid molecule that includes a target sequence. In such instances,
the target-specific primer and target sequence are described as
"corresponding" to each other. In some embodiments, the
target-specific primer is capable of hybridizing to at least a
portion of its corresponding target sequence (or to a complement of
the target sequence); such hybridization can optionally be
performed under standard hybridization conditions or under
stringent hybridization conditions. In some embodiments, the
target-specific primer is not capable of hybridizing to the target
sequence, or to its complement, but is capable of hybridizing to a
portion of a nucleic acid strand including the target sequence, or
to its complement. In some embodiments, a forward target-specific
primer and a reverse target-specific primer define a
target-specific primer pair that can be used to amplify the target
sequence via template-dependent primer extension. Typically, each
primer of a target-specific primer pair includes at least one
sequence that is substantially complementary to at least a portion
of a nucleic acid molecule including a corresponding target
sequence but that is less than 50% complementary to at least one
other target sequence in the sample. In some embodiments,
amplification can be performed using multiple target-specific
primer pairs in a single amplification reaction, wherein each
primer pair includes a forward target-specific primer and a reverse
target-specific primer, each including at least one sequence that
substantially complementary or substantially identical to a
corresponding target sequence in the sample, and each primer pair
having a different corresponding target sequence.
[0043] Tumor mutation load (TML) is a measure of the number of
mutations within a tumor genome, defined as the total number of
mutations per coding area of a tumor genome. Recent studies have
shown tumor mutation load to be a sensitive marker that can help
predict responses to certain cancer immunotherapies.
Immunotherapies have shown anti-cancer effects in melanoma,
non-small-cell lung carcinoma (NSCLC), and bladder cancer, among
other cancers. High tumor mutation load is associated with positive
responses from immune checkpoint inhibitors. Hence high mutation
load of a tumor may act as a predictive biomarker for
immunotherapy. However, existing methods to estimate tumor mutation
load have large input DNA and extensive infrastructure requirements
and are associated with delays due to shipping precious biopsy
samples to central laboratories.
[0044] In some embodiments, a targeted panel with low sample input
requirements may be used to estimate mutation load in a tumor
sample. A targeted panel for tumor mutation load, or TML panel,
provides a viable alternative to whole exome sequencing (WES). In
some embodiments, the targeted panel may comprise the Comprehensive
Cancer Panel (CCP) available from Thermo Fisher Scientific (SKU
4477685). The CCP interrogates 409 cancer genes, such as oncogenes
and tumor suppressor genes, using highly multiplexed amplification
with 4 pools of primer pairs that are targeted to the panel genes.
In some embodiments, the CCP may be modified to function with two
combined pools instead of four pools to reduce DNA sample size.
Removing the overlapping primers in the combined pools may reduce
number of primers in the modified CCP panel to produce a targeted
panel for TML including the same genes as the CCP. The targeted
panel interrogates 409 key cancer genes covering approximately 1.7
megabases (Mb) of genomic space. In some embodiments, the workflow
may require up to 20 ng DNA from formalin-fixed paraffin-embedded
(FFPE) or other sample types. In other embodiments, the workflow
may use about 1 ng to about 40 ng sample DNA. In other embodiments,
the workflow may use about 1 ng to about 20 ng or about 10 ng to
about 20 ng sample DNA. The embodiments described herein do not
require analysis of a matched normal sample to estimate the tumor
mutation load.
[0045] In some embodiments, the panel may comprise the Oncomine
Comprehensive Assay v3 (OCAv3) available from Thermo Fisher
Scientific (SKU A35806 or SKU A36111). The OCAv3 panel interrogates
161 cancer-related genes and enables detection of SNVs (single
nucleotide variants), CNVs (copy number variants), gene fusions and
indels using primer pairs targeted to the genes of the panel. In
some embodiments, the panel may comprise a custom panel or other
targeted panel of cancer driver or other genes associated with
cancer.
[0046] FIG. 1 is a block diagram of a method of detecting tumor
mutation load, according to an exemplary embodiment. In the variant
calling step 102, a processor receives aligned sequence reads
resulting from targeted sequencing of a tumor sample. The aligned
sequence reads can be retrieved from a file using a BAM file
format, for example. The aligned sequence reads may correspond to a
plurality of targeted locations in the tumor sample genome. The
variant calling step 102 may be configured by one or more variant
caller parameters. In some embodiments, variant caller parameters
may include parameters for minimum allele frequency, strand bias
and data quality stringency. The minimum allele frequency parameter
sets the minimum observed allele frequency required for a
non-reference variant call. The strand bias indicates a relative
proportion of variant reads in forward and reverse directional
sequencing. The strand bias parameter sets a maximum level for
strand bias such that a variant is not called if the proportion of
variant reads observed in one of the directional sequencings
exceeds the strand bias parameter. The data quality stringency
parameter sets a threshold for read quality required to make a
variant call. In some embodiments, the variant caller parameters
for single nucleotide variant (SNV) detection may be set to the
values given in Table 1.
TABLE-US-00001 TABLE 1 Variant Caller Parameter Value Range Minimum
Allele Frequency 0.05 or 0.1 0.001 to 0.15 Strand Bias 0.9 0.54 to
0.95 Data Quality Stringency 20 5 to 25
[0047] In some embodiments, variant caller parameters may include a
minimum coverage parameter, or minimum read depth parameter, that
sets a minimum coverage required for a variant to be called. The
minimum coverage parameter may be set to levels to reduce C>T or
G>A type nonsystematic noise. The minimum coverage parameter may
be set in a range from 20 to 60. The minimum coverage parameter of
gives a 10% level of detection (LOD) and minimum coverage parameter
of 60 gives a 5% level of LOD.
[0048] In some embodiments the aligned sequence reads are provided
by the mapping engine 308 described with respect to FIG. 15. In
some embodiments the variant calling step 102 may be implemented by
the variant calling engine 310 described with respect to FIG. 15.
In some embodiments, the variant detection methods for use with the
present teachings may include one or more features described in
U.S. Pat. Appl. Publ. No. 2013/0345066, published Dec. 26, 2013,
U.S. Pat. Appl. Publ. No. 2014/0296080, published Oct. 2, 2014, and
U.S. Pat. Appl. Publ. No. 2014/0052381, published Feb. 20, 2014,
each of which incorporated by reference herein in its entirety. In
some embodiments, other variant detection methods may be used. In
various embodiments, a variant caller can be configured to
communicate variants called for a sample genome as a *.vcf, *.gff,
or *.hdf data file. The called variant information can be
communicated using any file format as long as the called variant
information can be parsed and/or extracted for analysis.
[0049] Returning to FIG. 1, in the variant annotating step 104, a
processor annotates the detected variants with information
associated with the respective variants from one or more population
databases. In some embodiments, the annotation information may
include the minor allele frequency (MAF) of the variant. The
population database may provide public annotation information
content or proprietary annotation information content. For example,
publicly available population databases include: 5000 exomes--NHLBI
Exome Sequencing Project (http://evs.gs.washington.edu/EVS/), 1000
genomes--International Genome Sample Resource (IGSR)
(http://www.internationalgenome.org/home) and ExAC--Exome
Aggregation Consortium (http://exac.broadinstitute.org) and UCSC
common SNPs (https://genome.ucsc.edu/). Annotation information from
other population databases in addition to or in place of these
databases may be used. It may be understood that as genetic
information resources develop new and more extensive databases may
become available.
[0050] In some embodiments the annotating step 106 may be
implemented in the annotator component 314 and the population
database information may be stored in annotations data store 324
described with respect to FIG. 15. In some embodiments, the
annotation methods for use with the present teachings may include
one or more features described in U.S. Pat. Appl. Publ. No.
2016/0026753, published Jan. 28, 2016, incorporated by reference
herein in its entirety.
[0051] In the filtering step 106, the processor applies a rule set
to retain somatic variants and remove germ-line variants from the
detected variants. In some embodiments, a filter rule set is
applied to each detected variant and includes at least some of the
rules listed in Table 2.
TABLE-US-00002 TABLE 2 Filter Rule 1. Retain SNVs; optionally
filter out other variant types, such as indels and MNVs 2. Filter
out SNVs inside homopolymers with lengths greater than 7. 3. Retain
variants found in 1000 genomes with MAF in a given MAF range;
filter out variants outside the MAF range. 4. Retain variants found
in 5000 exomes with MAF in a given MAF range; filter out variants
outside the MAF range. 5. Retain variants found in ExAC with MAF in
a given MAF range; filter out variants outside the MAF range. 6.
Filter out variants found in UCSC common SNPs.
[0052] In some embodiments, a particular type of variant is
retained, such as SNVs only, for further analysis while other types
of variants are filtered out. Optionally, other types of variants,
such as indels, may be retained for further analysis. In some
embodiments, variants in regions with homopolymer lengths greater
than 7 are filtered out to mitigate lower accuracy in base calling
for long homopolymers. In filter rules 3, 4 and 5, detected
variants are retained if the MAF indicated by the population
database is within a given MAF range. The MAF is included in the
annotation information associated with the detected variants by the
annotating step 104. In a preferred embodiment, the MAF range is [0
10.sup.-6], or MAF is less than or equal to 10.sup.-6. In some
embodiments, the MAF range may be [0 0.001], [0 0.002] or [0 0.01].
The MAF ranges may be the same or different for the population
databases, such as the 1000 genomes, 5000 exomes and ExAC
databases. In filter rule 6, variants found in the UCSC common SNPs
database are filtered out. The filter rule set applied to the
detected variants removes the germ-line variants and retains the
somatic variants to produce identified somatic variants.
[0053] In the counting step 108, the processor counts the
identified somatic variants to produce a somatic mutation count. In
the determining step 110, the processor determines the covered
regions of the aligned sequence reads where the coverage of a given
base position is at least a threshold coverage. In some
embodiments, the threshold coverage may be in a range of 20 to 60
sequence reads. The threshold coverage of 20 corresponds to a
workflow for a 10% LOD. The threshold coverage of 60 corresponds to
a workflow for a 5% LOD. The processor counts the number of bases
in the covered regions to produce the covered base count in
megabases (Mb). In the calculating step 112, the processor divides
the somatic mutation count by the covered base count to form an
estimate of the tumor mutation load in number of somatic mutations
per Mb for the tumor sample genome. The tumor mutation load
estimate may be provided in a report to the user.
[0054] FIGS. 2A-2B, 3A-3B and 4A-4B show examples of results before
and after applying the filter rule set 106 described with respect
to FIG. 1. FIGS. 2A, 3A are 4A show histogram plots of the number
of variants per allele ratio including all detected variants, both
germ-line and somatic variants. The allele ratio of an allele is
the ratio of number of reads supporting this allele to the total
number of reads at the respective position. A cluster of detected
variants near an allele ratio of 0.5 may indicate germ-line alleles
from one parent. A cluster of detected variants near an allele
ratio of 1.0 may indicate germ-line alleles from both parents.
FIGS. 2B, 3B are 4B show histogram plots of the number of variants
per allele ratio after applying the filter rule set to remove the
germ-line variants by the filtering step 106. Numbers of variants
are significantly reduced, as shown in FIGS. 2B, 3B and 4B and in
Table 3. Table 3 also gives the results on somatic mutations per
megabase, or mutation load per megabase.
TABLE-US-00003 TABLE 3 NO. OF NO. OF VARIANTS VARIANTS SOMATIC
FIGURE BEFORE AFTER MUTATIONS/ NO. FILTER 106 FILTER 106 MB 2A
(BEFORE); 3,706 113 52.24 2B (AFTER) 3A (BEFORE); 1,057 120 73.14
3B (AFTER) 4A (BEFORE); 1,111 113 68.84 4B (AFTER)
[0055] To test the effectiveness of the tumor only analysis in
counting somatic variants, mutation counts resulting from of tumor
only analysis and matched tumor-normal analysis were compared.
FIGS. 5 and 6 shows examples of comparisons of somatic SNV mutation
counts resulting from analysis of tumor sample only and analysis of
matched tumor-normal samples. The somatic mutation counts in FFPE
tumor only samples determined using the method described with
respect to FIG. 1 were highly concordant with somatic mutation
counts determined by matched tumor-normal sequencing. Mutation
counts from tumor only analysis strongly correlate with those of
tumor-normal analysis. For the results of FIG. 5, the coefficient
of determination, r2=0.80. For the results of FIG. 6, the
correlation coefficient, r=0.9233.
[0056] FIG. 7 shows the results of testing the reproducibility of
the somatic mutation counts. The results show that the somatic
mutation loads estimated using the method described with respect to
FIG. 1 were highly reproducible. Sample types included cell lines,
FFPE and fresh frozen (FF) lung samples.
[0057] Cell line samples were obtained from NIGMS Human Genetic
Cell Repository at the Coriell Institute for Medical Research. FFPE
and FF samples were obtained from Biochain, Bioreclamation and
Conversant. Control samples were obtained from Acrometrix.
[0058] In silico analyses show that the targeted panel correlates
well with whole exome sequencing results for mutation counts. A
somatic variant dataset of WES data was derived from the COSMIC v80
database containing 21,056 exomes derived from 22 major cancer
types (http://cancer.sanger.ac.uk/cosmic). Somatic mutations in the
somatic variant dataset were restricted to those covered by the
targeted panel for TML. FIG. 8 shows a plot comparing the mutation
count through the targeted panel for TML versus the mutation count
through WES. The mutation counts plotted represent cancer types,
including bladder cancer, brain and central nervous system (CNS)
cancer, breast cancer, cervical cancer, colorectal cancer,
endometrial cancer, esophageal cancer, gastric cancer, head and
neck cancer, kidney cancer, leukemia, liver cancer, lung cancer,
lymphoma, melanoma, myeloma, other cancer, ovarian cancer,
pancreatic cancer, prostate cancer, sarcoma and thyroid cancer.
Somatic mutation counts in WES strongly correlated with mutation
counts in the targeted panel for TML with r2=0.968.
[0059] FIGS. 9A-9D show plots comparing the mutation count through
the targeted panel for TML versus the mutation count through WES
for single cancer types extracted from FIG. 8. FIG. 9A shows the
comparison of mutation counts between the targeted panel and WES
for melanoma, where r2=0.976. FIG. 9B shows the comparison of
mutation counts between the targeted panel and WES for lung cancer,
where r2=0.935. FIG. 9C shows the comparison of mutation counts
between the targeted panel and WES for colorectal cancer, where
r2=0.975. FIG. 9D shows the comparison of mutation counts between
the targeted panel and WES for endometrial cancer, where r2=0.995.
The results described with respect to FIG. 8 and FIGS. 9A-9D show
that the targeted panel is suitable for TML estimation.
[0060] Higher somatic mutation counts were associated with clinical
response to immune checkpoint blockade inhibitors in studies by
Rizvi et al. (2016 Science. 348:124-128) and Snyder et al. (2014 N
Eng J Med. 371:2189-2199). FIGS. 10A-10B and 11A-11B show an in
silico analysis of the results reported in the Rizvi and Snyder
studies using whole exome sequencing (WES). FIGS. 10A and 11A show
box plots for the WES TML counts versus clinical response for
results by Rizvi and results by Snyder, respectively. To compare
with the targeted panel, the somatic mutation counts intersecting
with the targeted panel including 409 genes were determined and
plotted. FIGS. 10B and 11B show box plots for the somatic mutation
counts intersecting with the targeted panel versus clinical
response. The results suggest the targeted panel is sufficiently
large to predict potential clinical outcome. P-values were
determined using the Mann-Whitney Exact test with no
assumptions.
[0061] High mutation load correlates with microsatellite
instability (MSI) in colorectal cancer. Thus MSI status can be used
for benchmarking. A batch of 7 colorectal cancer (CRC) tumor
samples in which three had known MSI high status and four had known
MSI low status were tested using the TML detection method described
with respect to FIG. 1. FIG. 12 shows the mutation counts per Mb
for MSI high and MSI low samples. MSI high status correlates to
high mutation count per Mb in CRC tumor sample. The results show
that the TML detection method successfully stratified MSI high and
MSI low samples based on mutation counts per Mb.
[0062] In some embodiments, sources of DNA damage related to the
somatic variants identified by the filtering step 106 may be
provided in a report to the user. Sources of DNA damage may
include, but are not limited to, ultraviolet (UV) light, tobacco
smoke, FFPE deamination and spontaneous deamination of
5-methylcytosine. Table 4 lists the types of somatic mutations
consistent with damage from these sources.
TABLE-US-00004 TABLE 4 SOURCE OF DAMAGE TYPES OF SOMATIC MUTATIONS
(1) Spontaneous deamination High C > T at CpG of
5-methylcytosine (2) UV damage High C > T at CpC, CpC, TpC, T
> A and T > C (3) Smoking damage High C > A (4) FFPE
processing damage High C > T (site independent)
[0063] The references for the relationships given in Table 4 are
(1) Alexandrov L B et al. Nature, 2013; (2) Hayward N K et al.
Nature, 2017; (3) Alexandrov L B et al. Cancer Etiology, 2016; and
(4) Wong S Q et al. BMC Medical Genomics, 2014.
[0064] In some embodiments, the processor may calculate the
percentages of the somatic mutations identified by the filtering
step 106 that match the types of somatic mutations consistent with
the various sources of damage. Table 5 gives an example of
percentages calculated for identified somatic variants matching the
types of somatic mutations given in Table 4. "Rest" is for those
identified somatic variants that did not match any of the types of
somatic mutations given in Table 4.
TABLE-US-00005 TABLE 5 TYPES OF SOMATIC FIG. 13 MUTATIONS
PERCENTAGE SECTOR T > A and T > C 54.2% 1304 Rest 20.8% 1305
C > A 16.7% 1303 C > T at NCC, 4.2% 1302 CC[ACT], TC[ACT] C
> T at [AG]CG 4.2% 1301
[0065] FIG. 13 is a pie chart for the pattern of somatic mutations
for the example of percentage values given in Table 4. The sectors
1301, 1302, 1303, 1304 and 1305 in the pie chart are noted in Table
4. In some embodiments, the pie chart of FIG. 13 and information of
Tables 4 and 5 may be included in a report for the user.
[0066] In some embodiments, the somatic mutations identified by the
filtering step 106 may be further analyzed to produce various
graphs, pie charts and histograms for a report. For example, the
percentages of identified somatic mutations having certain types of
substitutions may be calculated and displayed in a pie chart.
Examples of types of substitutions include C>A, C>G, C>T,
T>A, T>C and T>G.
[0067] The targeted panel and method for estimating tumor mutation
load described herein provide improvements to the technology over
whole exome sequencing (WES). Sequence assembly methods must be
able to assemble and/or map a large number of reads efficiently,
such as by minimizing use of computational resources. For example,
the sequencing of a human size genome can result in tens or
hundreds of millions of reads that need to be assembled before they
can be further analyzed. Computer processing of the nucleic acid
sequence reads from targeted sequencing reduces computational
requirements and memory requirements versus processing for WES
data. For WES, 30 Mb of the tumor genome would be covered. The data
resulting from the nucleic acid sequence reads of the 30 Mb would
require computations to detect variants and storage. In comparison,
the targeted panel that covers approximately 1.7 Mb of the tumor
genome would require substantially fewer computations for detecting
variants and substantially less memory for storage of the nucleic
acid sequence reads and variant data.
[0068] The targeted panel and method for estimating tumor mutation
load for a tumor only sample described herein provide improvements
to the technology over matched tumor-normal sample processing. In
some cases, a matched normal sample for the tumor sample may not be
available. When the matched normal sample is available, detecting
variants in the nucleic acid sequence reads from the normal sample
require at least the same amount of processing as for the tumor
sample, thereby at least doubling the computations and memory
requirements.
[0069] According to an exemplary embodiment, there is provided a
method of analyzing a tumor sample genome for a mutation load,
including the following steps: (1) detecting variants in a
plurality of nucleic acid sequence reads to produce a plurality of
detected variants, wherein the nucleic acid sequence reads
correspond to a plurality of targeted locations in the tumor sample
genome, wherein the detected variants include somatic variants and
germ-line variants; (2) annotating one or more detected variants of
the plurality of detected variants with an annotation information
from one or more population databases, wherein the population
databases include information associated with variants in a
population, wherein the annotation information includes a minor
allele frequency associated with a given variant; (3) filtering the
plurality of detected variants, wherein the filtering applies a
rule set to the detected variants to retain the somatic variants,
the rule set including retaining the detected variants whose minor
allele frequency (MAF) is within a MAF range, wherein the filtering
produces identified somatic variants; (4) counting the identified
somatic variants to give a number of somatic variants; (5)
determining a number of bases in covered regions of the targeted
locations in the tumor sample genome; and (6) calculating a number
of somatic variants per megabase by dividing the number of
identified somatic variants by the number of bases in the covered
regions to produce the mutation load for the tumor sample genome.
The MAF range may be from 0 to 10.sup.-6. The population databases
may include one or more of a 1000 genomes database, a 5000 exomes
database and an Exome Aggregation Consortium (ExAC) database. The
rule set for the filtering step may further comprise retaining the
detected variants that are single nucleotide variants (SNVs). The
rule set may further comprise retaining the detected variants that
are SNVs, insertion variants and deletion variants (indels). The
rule set may further comprise removing the detected variants that
are SNVs corresponding to SNPs in the UCSC Common SNP database. The
rule set may further comprise removing the detected variants in
regions having homopolymer lengths greater than seven. The
detecting variants may be configured by variant caller parameters,
including a minimum allele frequency parameter, a strand bias
parameter and a data quality stringency parameter. The minimum
allele frequency parameter may be in a range from 0.001 to 0.15,
the strand bias parameter may be in a range from 0.54 to 0.95 and
the data quality stringency parameter is in a range from 5 to 25.
The method may further comprise associating sources of somatic
mutation damage with the identified somatic variants.
[0070] According to an exemplary embodiment, there is provided a
system for analyzing a tumor sample genome for a mutation load,
comprising a processor and a data store communicatively connected
with the processor, the processor configured to perform the steps
including: detecting variants in a plurality of nucleic acid
sequence reads to produce a plurality of detected variants, wherein
the nucleic acid sequence reads correspond to a plurality of
targeted locations in the tumor sample genome, wherein the detected
variants include somatic variants and germ-line variants;
annotating one or more detected variants of the plurality of
detected variants with an annotation information from one or more
population databases stored in the data store, wherein the
population databases include information associated with variants
in a population, wherein the annotation information includes a
minor allele frequency associated with a given variant; filtering
the plurality of detected variants, wherein the filtering applies a
rule set to the detected variants to retain the somatic variants,
the rule set including retaining the detected variants whose minor
allele frequency (MAF) is within a MAF range, wherein the filtering
produces identified somatic variants; counting the identified
somatic variants to give a number of somatic variants; determining
a number of bases in covered regions of the targeted locations in
the tumor sample genome; and calculating a number of somatic
variants per megabase by dividing the number of identified somatic
variants by the number of bases in the covered regions to produce
the mutation load for the tumor sample genome. The MAF range may be
from 0 to 10.sup.-6. The population databases may include one or
more of a 1000 genomes database, a 5000 exomes database and an
Exome Aggregation Consortium (ExAC) database. The rule set for the
filtering step may further comprise retaining the detected variants
that are single nucleotide variants (SNVs). The rule set may
further comprise retaining the detected variants that are SNVs,
insertion variants and deletion variants (indels). The rule set may
further comprise removing the detected variants that are SNVs
corresponding to SNPs in the UCSC Common SNP database. The rule set
may further comprise removing the detected variants in regions
having homopolymer lengths greater than seven. The detecting
variants may be configured by variant caller parameters, including
a minimum allele frequency parameter, a strand bias parameter and a
data quality stringency parameter. The minimum allele frequency
parameter may be in a range from 0.001 to 0.15, the strand bias
parameter may be in a range from 0.54 to 0.95 and the data quality
stringency parameter is in a range from 5 to 25. The steps may
further comprise associating sources of somatic mutation damage
with the identified somatic variants.
[0071] According to an exemplary embodiment, there is provided a
non-transitory machine-readable storage medium comprising
instructions which, when executed by a processor, cause the
processor to perform a method for analyzing a tumor sample genome
for a mutation load, including: (1) detecting variants in a
plurality of nucleic acid sequence reads to produce a plurality of
detected variants, wherein the nucleic acid sequence reads
correspond to a plurality of targeted locations in the tumor sample
genome, wherein the detected variants include somatic variants and
germ-line variants; (2) annotating one or more detected variants of
the plurality of detected variants with an annotation information
from one or more population databases, wherein the population
databases include information associated with variants in a
population, wherein the annotation information includes a minor
allele frequency associated with a given variant; (3) filtering the
plurality of detected variants, wherein the filtering applies a
rule set to the detected variants to retain the somatic variants,
the rule set including retaining the detected variants whose minor
allele frequency (MAF) is within a MAF range, wherein the filtering
produces identified somatic variants; (4) counting the identified
somatic variants to give a number of somatic variants; (5)
determining a number of bases in covered regions of the targeted
locations in the tumor sample genome; and (6) calculating a number
of somatic variants per megabase by dividing the number of
identified somatic variants by the number of bases in the covered
regions to produce the mutation load for the tumor sample genome.
The MAF range may be from 0 to 10'. The population databases may
include one or more of a 1000 genomes database, a 5000 exomes
database and an Exome Aggregation Consortium (ExAC) database. The
rule set for the filtering step may further comprise retaining the
detected variants that are single nucleotide variants (SNVs). The
rule set may further comprise retaining the detected variants that
are SNVs, insertion variants and deletion variants (indels). The
rule set may further comprise removing the detected variants that
are SNVs corresponding to SNPs in the UCSC Common SNP database. The
rule set may further comprise removing the detected variants in
regions having homopolymer lengths greater than seven. The
detecting variants may be configured by variant caller parameters,
including a minimum allele frequency parameter, a strand bias
parameter and a data quality stringency parameter. The minimum
allele frequency parameter may be in a range from 0.001 to 0.15,
the strand bias parameter may be in a range from 0.54 to 0.95 and
the data quality stringency parameter is in a range from 5 to 25.
The steps may further comprise associating sources of somatic
mutation damage with the identified somatic variants.
[0072] In various embodiments, nucleic acid sequence data can be
generated using various techniques, platforms or technologies,
including, but not limited to: capillary electrophoresis,
microarrays, ligation-based systems, polymerase-based systems,
hybridization-based systems, direct or indirect nucleotide
identification systems, pyrosequencing, ion- or pH-based detection
systems, electronic signature-based systems, fluorescent-based
detection systems, single molecule methods, etc.
[0073] Various embodiments of nucleic acid sequencing platforms,
such as a nucleic acid sequencer, can include components as
displayed in the block diagram of FIG. 14. According to various
embodiments, sequencing instrument 200 can include a fluidic
delivery and control unit 202, a sample processing unit 204, a
signal detection unit 206, and a data acquisition, analysis and
control unit 208. Various embodiments of instrumentation, reagents,
libraries and methods used for next generation sequencing are
described in U.S. Patent Application Publication No. 2009/0127589
and No. 2009/0026082. Various embodiments of instrument 200 can
provide for automated sequencing that can be used to gather
sequence information from a plurality of sequences in parallel,
such as substantially simultaneously.
[0074] In various embodiments, the fluidics delivery and control
unit 202 can include reagent delivery system. The reagent delivery
system can include a reagent reservoir for the storage of various
reagents. The reagents can include RNA-based primers,
forward/reverse DNA primers, oligonucleotide mixtures for ligation
sequencing, nucleotide mixtures for sequencing-by-synthesis,
optional ECC oligonucleotide mixtures, buffers, wash reagents,
blocking reagent, stripping reagents, and the like. Additionally,
the reagent delivery system can include a pipetting system or a
continuous flow system which connects the sample processing unit
with the reagent reservoir.
[0075] In various embodiments, the sample processing unit 204 can
include a sample chamber, such as flow cell, a substrate, a
micro-array, a multi-well tray, or the like. The sample processing
unit 204 can include multiple lanes, multiple channels, multiple
wells, or other means of processing multiple sample sets
substantially simultaneously. Additionally, the sample processing
unit can include multiple sample chambers to enable processing of
multiple runs simultaneously. In particular embodiments, the system
can perform signal detection on one sample chamber while
substantially simultaneously processing another sample chamber.
Additionally, the sample processing unit can include an automation
system for moving or manipulating the sample chamber.
[0076] In various embodiments, the signal detection unit 206 can
include an imaging or detection sensor. For example, the imaging or
detection sensor can include a CCD, a CMOS, an ion sensor, such as
an ion sensitive layer overlying a CMOS, a current detector, or the
like. The signal detection unit 206 can include an excitation
system to cause a probe, such as a fluorescent dye, to emit a
signal. The expectation system can include an illumination source,
such as arc lamp, a laser, a light emitting diode (LED), or the
like. In particular embodiments, the signal detection unit 206 can
include optics for the transmission of light from an illumination
source to the sample or from the sample to the imaging or detection
sensor. Alternatively, the signal detection unit 206 may not
include an illumination source, such as for example, when a signal
is produced spontaneously as a result of a sequencing reaction. For
example, a signal can be produced by the interaction of a released
moiety, such as a released ion interacting with an ion sensitive
layer, or a pyrophosphate reacting with an enzyme or other catalyst
to produce a chemiluminescent signal. In another example, changes
in an electrical current can be detected as a nucleic acid passes
through a nanopore without the need for an illumination source.
[0077] In various embodiments, data acquisition analysis and
control unit 208 can monitor various system parameters. The system
parameters can include temperature of various portions of
instrument 200, such as sample processing unit or reagent
reservoirs, volumes of various reagents, the status of various
system subcomponents, such as a manipulator, a stepper motor, a
pump, or the like, or any combination thereof.
[0078] It will be appreciated by one skilled in the art that
various embodiments of instrument 200 can be used to practice
variety of sequencing methods including ligation-based methods,
sequencing by synthesis, single molecule methods, nanopore
sequencing, and other sequencing techniques.
[0079] In various embodiments, the sequencing instrument 200 can
determine the sequence of a nucleic acid, such as a polynucleotide
or an oligonucleotide. The nucleic acid can include DNA or RNA, and
can be single stranded, such as ssDNA and RNA, or double stranded,
such as dsDNA or a RNA/cDNA pair. In various embodiments, the
nucleic acid can include or be derived from a fragment library, a
mate pair library, a ChIP fragment, or the like. In particular
embodiments, the sequencing instrument 200 can obtain the sequence
information from a single nucleic acid molecule or from a group of
substantially identical nucleic acid molecules.
[0080] In various embodiments, sequencing instrument 200 can output
nucleic acid sequencing read data in a variety of different output
data file types/formats, including, but not limited to: *.fasta,
*.csfasta, *seq.txt, *qseq.txt, *.fastq, *.sff, *prb.txt, *.sms,
*srs and/or *.qv.
[0081] FIG. 15 is a schematic diagram of a system for annotating
genomic variants, in accordance with various embodiments.
[0082] As depicted herein, annotation system 300 can include a
nucleic acid sequence analysis device 304 (for example, nucleic
acid sequencer, real-time/digital/quantitative PCR instrument,
microarray scanner, etc.), an analytics computing
server/node/device 302, a display 338 and/or a client device
terminal 336, and one or more public 330 and proprietary 332
annotations content sources.
[0083] In various embodiments, the analytics computing
server/node/device 302 can be communicatively connected to the
nucleic acid sequence analysis device 304, client device terminal
336, public annotations content source 330 and/or proprietary
annotations content source 332 via a network connection 334 that
can be either a "hardwired" physical network connection (for
example, Internet, LAN, WAN, VPN, etc.) or a wireless network
connection (for example, Wi-Fi, WLAN, etc.).
[0084] In various embodiments, the analytics computing
device/server/node 302 can be a workstation, mainframe computer,
distributed computing node (part of a "cloud computing" or
distributed networking system), personal computer, mobile device,
etc. In various embodiments, the nucleic acid sequence analysis
device 304 can be a nucleic acid sequencer,
real-time/digital/quantitative PCR instrument, microarray scanner,
etc. It should be understood, however, that the nucleic acid
sequence analysis device 304 can essentially be any type of
instrument that can generate nucleic acid sequence data from
samples obtained from an individual 306.
[0085] The analytics computing server/node/device 302 can be
configured to host a mapping engine 308, a variant calling engine
310, a decision support module 312 and a reporter module 316.
[0086] The mapping engine 308 can be configured to align or map a
query nucleic acid sequence read to a reference sequence.
Generally, the length of the sequence read is substantially less
than the length of the reference sequence. In reference sequence
mapping/alignment, sequence reads can be assembled against an
existing backbone sequence (for example, reference sequence, etc.)
to build a sequence that is similar but not necessarily identical
to the backbone sequence. Once a backbone sequence is found for an
organism, comparative sequencing or re-sequencing can be used to
characterize the genetic diversity within the organism's species or
between closely related species. In various embodiments, the
reference sequence can be a whole/partial genome, whole/partial
exome, whole/partial transcriptome, etc.
[0087] In various embodiments, the sequence read and reference
sequence can be represented as a sequence of nucleotide base
symbols in base space. In various embodiments, the sequence read
and reference sequence can be represented as one or more color
symbols in color space. In various embodiments, the sequence read
and reference sequence can be represented as nucleotide base
symbols with signal or numerical quantitation components in flow
space.
[0088] In various embodiments, the alignment of the sequence read
and reference sequence can include a limited number of mismatches
between the bases that comprise the sequence read and the bases
that comprise the reference sequence. Generally, at least a portion
of the sequence read can be aligned to a portion of the reference
sequence, such as a reference nuclear genome, a reference
mitochondrial genome, a reference prokaryotic genome, a reference
chloroplast genome, or the like, in order to minimize the number of
mismatches between the sequence fragment and the reference
sequence.
[0089] The variant calling engine 310 can be configured to receive
aligned sequence reads from the mapping engine 308 and analyze the
aligned sequence reads to detect and call or identify one or more
variants within the reads. Examples of variants that can be called
by a variant calling engine 310 include but are not limited to:
single nucleotide variants (SNV), single nucleotide polymorphisms
(SNP), nucleotide insertions or deletions (indels), copy number
variations (CNV) identification, inversion polymorphisms, and the
like.
[0090] The reporter module 316 can be in communications with the
decision support module 312 and be configured to generate a summary
report of the called genomic variants that have been annotated by
the annotator component 314 that can be part of the decision
support module 312.
[0091] The decision support module can include an annotator
component 314, a variome data store 322, an annotations data store
324, a filtering component 328 and/or an annotations importer
component 326. In various embodiments, the annotator component 314
can be in communication with the variant calling engine 310, the
variome data store 322 and/or the annotations data store 324. That
is, the annotator component 314 can request and receive data and
information (through, for example, data streams, data files, text
files, etc.) from variant calling engine 310, variome data store
322 and annotations data store 324. In various embodiments, the
variant calling engine 310 can be configured to communicate
variants called for a sample genome in various formats, such as,
but not limited to, variant call format (VCF), generic feature
format (GFF) heirachical data format (HDF), genome variation format
(GVF), or HL7 formatted data. It should be understood, however,
that the called variants can be communicated using any file format
where the called variant information can be parsed and/or extracted
for later processing/analysis.
[0092] The variome data store 322 can be configured to store the
variant calls received from the variant calling engine 310 and/or
the annotator component 314 in a format that is accessible for
mining.
[0093] That is, the called variant data can be maintained as a
database or instantiated in some other persistent (and queryable)
electronic form in the device memory (for example, hard drive, RAM,
ROM, etc.) of the analytics computing server/node/device 302. The
called variant data can be structured and use a common syntax and
semantic model throughout or include appropriate interpreters
between formats that allow for one-to-one mapping between terms and
data types. In various embodiments, the variome data store 322 can
be an indexed database table of variants. In particular
embodiments, the indexed database can be configured for fast
querying and filtering operations.
[0094] The annotations data store 324 can be in communications with
the annotations importer component 326 and be configured to store
data and information that can be used by the annotator component
314 to annotate the called variants. That is, the annotations data
store 324 can store annotation data and information that can be
relevant to the role that the called variant plays in the function,
such as at a chromosome level, gene level, a transcript level, a
protein level, or the like, (for example, functional type
annotations) and/or the biological impact (for example,
interpretive type annotations) of the called variants. In various
embodiments, functional type annotations can include, but are not
limited to: locus classification of the called variant, protein
function impact score of the called variant, amino acid changes
resulting from the called variant, gene/transcripts affected by the
called variant, etc. In various embodiments, interpretive type
annotations can include, but are not limited to: disease states or
susceptibility to a disease (for example, cancer, diabetes,
hypertension, heart disease, etc.) associated with the called
variant, impacts that the called variant has on a particular
therapeutic regimen (for example, drugs, surgical options, medical
device, psychiatric therapy, lifestyle changes, drug sensitivities,
etc.), presence of the variant on a list of annotated variants,
etc. For example, a SNP variant call can be annotated with
functional type annotations that point to the transcripts that the
called SNP impacts and interpretive type annotations that are
directed to diagnosing a particular disease state or a
susceptibility to a disease.
[0095] The annotations importer component 326 can be configured to
receive annotations content from one or more public 330 or
proprietary 332 annotations content sources and convert the
annotations content into a format that can be stored in the
annotations data store 324 and is accessible for mining. That is,
the annotations importer component 326 can convert annotations data
and/or information into a format that can be stored onto a database
or instantiated in some other persistent (and queryable) electronic
form in the device memory (for example, hard drive, RAM, ROM, etc.)
of the analytics computing server/node/device 302.
[0096] In various embodiments, annotations content can be manually
entered or uploaded by a user to the annotations importer component
326 via a computer readable storage medium that is communicatively
connected (for example, via a serial data bus connection, parallel
data bus connection, internet/intranet network connection, etc.) to
the analytics computing server/node/device 302. That is, a user can
selectively upload annotations content to the annotations data
store 324 depending on the requirements of the particular
application. Examples of computer readable medium include, but are
not limited to: hard drives, network attached storage (NAS),
read-only memory, random-access memory, CD-ROMs, CD-Rs, CD-RWs,
magnetic tapes, FLASH memory and other optical/non-optical data
storage devices.
[0097] In various embodiments, annotations content can be
automatically requested and sent from public 330 and/or proprietary
332 annotations content sources to the annotations importer
component 326 through the use of a data refresh executable or
script. That is, the annotations content in the annotations data
store 324 can be continuously refreshed as the public 330 and/or
proprietary 332 annotations content sources are updated with new or
modified annotations content.
[0098] In various embodiments, the annotator component 314 can
include a functional annotations engine 318 and interpretive
annotations engine 320.
[0099] The functional annotations engine 318 can be configured to
receive called variants from the variome data store 322, associate
one or more functional type annotations (stored in the annotations
data store 324) to the called variants and update the called
variant records in the variome data store 322 with the associated
functional type annotations. In various embodiments, the functional
annotations engine 318 can be configured to annotate all called
variants that fall within a block of overlapping transcripts (in
the sample genome) at the same time. That is, the functional
annotations engine 318 can group overlapping transcripts together
into a "gene block" and then annotate all variants in the gene
block together. The advantage here is that all called variants that
are potentially mutually interacting can be grouped and annotated
together to give researchers/clinicians greater insight into the
synergistic or antagonistic interplay between variants.
[0100] In various embodiments, the functional annotations engine
318 can be selectively configured to annotate only called variants
that fall within a coding region (for example, exons, codons) of
the sample genome being annotated. In various embodiments, the
functional annotations engine 318 can be selectively configured to
annotate only called variants that fall within an intragenic
region, such as an intron, of the sample genome being annotated. In
various embodiments, the functional annotations engine 318 can be
selectively configured to annotate only the called variants in the
intergenic region of the sample genome being annotated.
[0101] In various embodiments, the functional annotations engine
318 can receive the called variants in the form of a called variant
data file (for example, *.vcf or other file format), associate the
functional type annotations, and store the variants and annotations
to the variome data store 322. In various embodiments, the
functional annotations engine 318 can receive the called variants
as variant data (for example, variant base identity and genome
position, etc.), associates one or more functional type annotations
to the called variant and directly updates the called variant
record in the variome data store 322 with the associated functional
type annotations information. That is, the functional annotations
engine 318 can receive called variants directly from the variome
data store 322, annotate them and save them back on the variome
data store 322 or alternate data store.
[0102] The interpretive annotations engine 320 can be configured to
receive called variants from the variome data store 322, associate
one or more interpretive type annotations (stored in the
annotations data store 324) to the called variants and update the
called variant records in the variome data store 322 with the
associated interpretive type annotations.
[0103] In various embodiments, the interpretive annotations engine
320 receives the called variants in the form of a called variant
data file (for example, *.vcf or other file format), associate the
interpretive type annotations, and store the variants and
annotations to the variome data store 322. In various embodiments,
the interpretive annotations engine 318 receives the called
variants as variant data (for example, variant base identity and
genome position, etc.), associates one or more interpretive type
annotations to the called variant and directly updates the called
variant record in the variome data store 322 with the associated
interpretive type annotations information.
[0104] In various embodiments, the system can be configured to
automate the processing of sample data. For example, a workflow can
be selected to define how the data is processed by the mapping
engine 308, the variant calling engine 310, and the annotator
component 314. In particular embodiments, a workflow can be
selected when setting up the run on the nucleic acid sequence
analysis device 304 and the data can be automatically uploaded to
the analytics computing device 302. Additionally, the workflow can
be automatically launched when the data has been uploaded. In other
embodiments, the data can be uploaded, manually or automatically,
from the nucleic acid sequence analysis device 304 and the workflow
can be selected and launched manually. Generally, once the workflow
has been selected and launched, analysis can proceed from through
the mapping engine 308, the variant calling engine, 310, and the
annotator component 314 without further intervention by a user.
[0105] The filtering component 328 can be configured to allow a
user to set filter conditions to filter the called variants that
are included in the summary report generated by the reporter module
316. Examples of filter conditions include, but are not limited to,
filtering for: variants that are non-synonymous and fall within a
particular gene, variants that are associated with a particular
disease condition, variants that have a functional score of greater
or less than a selected value, novel variants that are not present
in a functional type annotations source, variants that fall in gene
panel regions (defined by user), etc. In various embodiments, the
filtering component 328 can utilize combinations of filters, such
as for example filtering for variants that fall within a particular
gene and have a functional score indicative of a significant
effect.
[0106] In various embodiments, the filtering component 328 can be
configured with a collection of filters to select for variants with
a high likelihood of having possible functional significance. For
example, the filtering component 328 can select for missense
mutations and nonsense mutations and exclude synonymous mutations.
Still further, the filtering component 328 can select for variants
that affect allele frequency. Also, the filtering component 328 may
select or exclude variants at positions of known significance, such
as positions known to have a high incidence of mutation in cancers,
positions with a low or high number of false positive variant
calls, positions known to have a minimal functional impact, or the
like.
[0107] In various embodiments, the variome data 322 and the
annotations data 324 stores can be combined into a single data
store configured to store both called variant data and variant
annotations information.
[0108] Client terminal 336 can be a thin client or thick client
computing device. In various embodiments, client terminal 336 can
have a web browser (for example, INTERNET EXPLORER.TM. FIREFOX.TM.,
SAFARI.TM., etc.) that can be used to communicate information to
and/or control the operation of the mapping engine 308, variant
calling engine 310, decision support module 312, annotator
component 314, filtering component 328, annotations importer
component 326, variome data store 322, annotations data store 324,
functional annotations engine 318 and/or interpretive annotations
engine 320 using a browser to control their function. For example,
the client terminal 336 can be used to configure the operating
parameters (for example, match scoring parameters, annotations
parameters, filtering parameters, data security and retention
parameters, etc.) of the various modules, depending on the
requirements of the particular application. Similarly, client
terminal 336 can also be configured to display the results of the
analysis performed by the decision support module 312 and the
nucleic acid sequencer 304.
[0109] It should be understood that the various data stores
disclosed as part of system 300 can represent hardware-based
storage devices (for example, hard drive, flash memory, RAM, ROM,
network attached storage, etc.) or instantiations of a database
stored on a standalone or networked computing device(s).
[0110] It should also be appreciated that the various data stores
and modules/engines shown as being part of the system 300 can be
combined or collapsed into a single module/engine/data store,
depending on the requirements of the particular application or
system architecture. Moreover, in various embodiments, the system
300 can comprise additional modules, engines, components or data
stores as needed by the particular application or system
architecture or to extend functionality.
[0111] In various embodiments, the system 300 can be configured to
process the nucleic acid reads in color space. In various
embodiments, system 300 can be configured to process the nucleic
acid reads in base space. In various embodiments, system 300 can be
configured to process the nucleic acid sequence reads in flow
space. It should be understood, however, that the system 300
disclosed herein can process or analyze nucleic acid sequence data
in any schema or format as long as the schema or format can convey
the base identity and position (or position range) of the nucleic
acid sequence within the reference sequence.
[0112] In various embodiments, the system 300 can be configured to
distinguish between positions with a called variant, positions that
have been called as reference, and positions with no call.
Positions with a called variant can include positions where
sufficient evidence was provided by the reads to indicate the
specimen sequence contains a variant. Positions that have been
called as reference can include positions where there is sufficient
evidence to support the conclusion that the specimen sequence is
substantially identical to the reference sequence at the position.
Positions with no call can include positions where there is
insufficient evidence to determine if the specimen sequence is the
same as or different from the reference sequence. For example,
positions with no call can include positions with low coverage,
positions with low base quality, or positions where the read
sequences indicate different bases with insufficient homogeneity to
determine the sequence with sufficient confidence. Generally,
positions with no call can be indicated as matching the reference
sequence and may be excluded from reporting of variants.
[0113] According to various exemplary embodiments, one or more
features of any one or more of the above-discussed teachings and/or
exemplary embodiments may be performed or implemented using
appropriately configured and/or programmed hardware and/or software
elements. Determining whether an embodiment is implemented using
hardware and/or software elements may be based on any number of
factors, such as desired computational rate, power levels, heat
tolerances, processing cycle budget, input data rates, output data
rates, memory resources, data bus speeds, etc., and other design or
performance constraints.
[0114] Examples of hardware elements may include processors,
microprocessors, input(s) and/or output(s) (I/O) device(s) (or
peripherals) that are communicatively coupled via a local interface
circuit, circuit elements (e.g., transistors, resistors,
capacitors, inductors, and so forth), integrated circuits,
application specific integrated circuits (ASIC), programmable logic
devices (PLD), digital signal processors (DSP), field programmable
gate array (FPGA), logic gates, registers, semiconductor device,
chips, microchips, chip sets, and so forth. The local interface may
include, for example, one or more buses or other wired or wireless
connections, controllers, buffers (caches), drivers, repeaters and
receivers, etc., to allow appropriate communications between
hardware components. A processor is a hardware device for executing
software, particularly software stored in memory. The processor can
be any custom made or commercially available processor, a central
processing unit (CPU), an auxiliary processor among several
processors associated with the computer, a semiconductor based
microprocessor (e.g., in the form of a microchip or chip set), a
macroprocessor, or generally any device for executing software
instructions. A processor can also represent a distributed
processing architecture. The I/O devices can include input devices,
for example, a keyboard, a mouse, a scanner, a microphone, a touch
screen, an interface for various medical devices and/or laboratory
instruments, a bar code reader, a stylus, a laser reader, a
radio-frequency device reader, etc. Furthermore, the I/O devices
also can include output devices, for example, a printer, a bar code
printer, a display, etc. Finally, the I/O devices further can
include devices that communicate as both inputs and outputs, for
example, a modulator/demodulator (modem; for accessing another
device, system, or network), a radio frequency (RF) or other
transceiver, a telephonic interface, a bridge, a router, etc.
[0115] Examples of software may include software components,
programs, applications, computer programs, application programs,
system programs, machine programs, operating system software,
middleware, firmware, software modules, routines, subroutines,
functions, methods, procedures, software interfaces, application
program interfaces (API), instruction sets, computing code,
computer code, code segments, computer code segments, words,
values, symbols, or any combination thereof. A software in memory
may include one or more separate programs, which may include
ordered listings of executable instructions for implementing
logical functions. The software in memory may include a system for
identifying data streams in accordance with the present teachings
and any suitable custom made or commercially available operating
system (O/S), which may control the execution of other computer
programs such as the system, and provides scheduling, input-output
control, file and data management, memory management, communication
control, etc.
[0116] According to various exemplary embodiments, one or more
features of any one or more of the above-discussed teachings and/or
exemplary embodiments may be performed or implemented using
appropriately configured and/or programmed non-transitory
machine-readable medium or article that may store an instruction or
a set of instructions that, if executed by a machine, may cause the
machine to perform a method and/or operations in accordance with
the exemplary embodiments. Such a machine may include, for example,
any suitable processing platform, computing platform, computing
device, processing device, computing system, processing system,
computer, processor, scientific or laboratory instrument, etc., and
may be implemented using any suitable combination of hardware
and/or software. The machine-readable medium or article may
include, for example, any suitable type of memory unit, memory
device, memory article, memory medium, storage device, storage
article, storage medium and/or storage unit, for example, memory,
removable or non-removable media, erasable or non-erasable media,
writeable or re-writeable media, digital or analog media, hard
disk, floppy disk, read-only memory compact disc (CD-ROM),
recordable compact disc (CD-R), rewriteable compact disc (CD-RW),
optical disk, magnetic media, magneto-optical media, removable
memory cards or disks, various types of Digital Versatile Disc
(DVD), a tape, a cassette, etc., including any medium suitable for
use in a computer. Memory can include any one or a combination of
volatile memory elements (e.g., random access memory (RAM, such as
DRAM, SRAM, SDRAM, etc.)) and nonvolatile memory elements (e.g.,
ROM, EPROM, EEROM, Flash memory, hard drive, tape, CDROM, etc.).
Moreover, memory can incorporate electronic, magnetic, optical,
and/or other types of storage media. Memory can have a distributed
architecture where various components are situated remote from one
another, but are still accessed by the processor. The instructions
may include any suitable type of code, such as source code,
compiled code, interpreted code, executable code, static code,
dynamic code, encrypted code, etc., implemented using any suitable
high-level, low-level, object-oriented, visual, compiled and/or
interpreted programming language.
[0117] According to various exemplary embodiments, one or more
features of any one or more of the above-discussed teachings and/or
exemplary embodiments may be performed or implemented at least
partly using a distributed, clustered, remote, or cloud computing
resource.
[0118] According to various exemplary embodiments, one or more
features of any one or more of the above-discussed teachings and/or
exemplary embodiments may be performed or implemented using a
source program, executable program (object code), script, or any
other entity comprising a set of instructions to be performed. When
a source program, the program can be translated via a compiler,
assembler, interpreter, etc., which may or may not be included
within the memory, so as to operate properly in connection with the
O/S. The instructions may be written using (a) an object oriented
programming language, which has classes of data and methods, or (b)
a procedural programming language, which has routines, subroutines,
and/or functions, which may include, for example, C, C++, R,
Pascal, Basic, Fortran, Cobol, Perl, Java, and Ada.
[0119] According to various exemplary embodiments, one or more of
the above-discussed exemplary embodiments may include transmitting,
displaying, storing, printing or outputting to a user interface
device, a computer readable storage medium, a local computer system
or a remote computer system, information related to any
information, signal, data, and/or intermediate or final results
that may have been generated, accessed, or used by such exemplary
embodiments. Such transmitted, displayed, stored, printed or
outputted information can take the form of searchable and/or
filterable lists of runs and reports, pictures, tables, charts,
graphs, spreadsheets, correlations, sequences, and combinations
thereof, for example.
[0120] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *
References