SYSTEMS AND METHODS TO DETECT RARE MUTATIONS AND COPY NUMBER VARIATION

• US 20150299812A1
• Filed: 05/14/2015
• Published: 10/22/2015
• Est. Priority Date: 09/04/2012
• Status: Active Grant

• Current Assignee: Guardant Health Inc.
• Original Assignee: Guardant Health Inc.
• Inventors: Talasaz, AmirAli
• Granted Patent: US 9,598,731 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

The present disclosure provides a system and method for the detection of rare mutations and copy number variations in cell free polynucleotides. Generally, the systems and methods comprise sample preparation, or the extraction and isolation of cell free polynucleotide sequences from a bodily fluid; subsequent sequencing of cell free polynucleotides by techniques known in the art; and application of bioinformatics tools to detect rare mutations and copy number variations as compared to a reference. The systems and methods also may contain a database or collection of different rare mutations or copy number variation profiles of different diseases, to be used as additional references in aiding detection of rare mutations, copy number variation profiling or general genetic profiling of a disease.

Claims

1. A method, comprising:

providing initial starting genetic material obtained from a bodily sample obtained from a subject;

converting double stranded polynucleotides from the initial starting genetic material into at least one set of non-uniquely tagged parent polynucleotides, wherein each polynucleotide in a set is mappable to a reference sequence; and

for each set of tagged parent polynucleotides;

i. amplifying the tagged parent polynucleotides in the set to produce a corresponding set of amplified progeny polynucleotides;

ii. sequencing the set of amplified progeny polynucleotides to produce a set of sequencing reads;

iii. collapsing the set of sequencing reads to generate a set of consensus sequences, wherein collapsing uses sequence information from a tag and at least one of;

sequence information at a beginning region of a sequence read, an end region of the sequence read and length of the sequence read, each consensus sequence corresponding to a unique polynucleotide among the set of tagged parent polynucleotides; and

ii. analyzing the set of consensus sequences for each set of tagged parent molecules separately or in combination.

2. The method of claim 1, wherein analyzing comprises detecting mutations, indels, copy number variations, transversions, translocations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, abnormal changes in nucleic acid methylation infection or cancer.

3. The method of claim 1, wherein analyzing comprises detecting the presence of sequence variations in the set of consensus sequences compared with the reference sequence.

4. The method of claim 3, wherein the mappable position in the reference sequence is the locus of a tumor marker, and wherein detecting the presence of sequence variations comprises detecting the tumor marker in the set of consensus sequences.

5. The method of claim 4, further comprising detecting the presence of sequence variations for a panel of oncogenes.

6. The method of claim 1, wherein the at least one set of non-uniquely tagged parent polynucleotides is a plurality of sets, and wherein the analyzing comprises normalizing copy number variation of consensus sequences between different mappable reference sequences.

7. The method of claim 1, wherein collapsing also uses information about length of each sequence read.

8. The method of claim 1, comprising filtering out reads that fail to meet a set threshold.

9. The method of claim 1, wherein the initial starting genetic material is tagged with between 5 and 100 different identifiers.

10. The method of claim 1, wherein the initial starting genetic material comprises between about 10 ng and 10 μ

g of fragmented polynucleotides.

11. The method of claim 1, wherein the initial starting genetic material comprises no more than 100 ng of polynucleotides.

12. The method of claim 1, wherein converting comprises blunt-end ligation or sticky end ligation.

13. The method of claim 1, further comprising bottlenecking the initial starting genetic material prior to converting.

14. The method of claim 1, further comprising converting the initial starting genetic material into tagged parent polynucleotides with a conversion efficiency of at least 10%.

15. The method of claim 1, further comprising converting the initial starting genetic material into tagged parent polynucleotides with a conversion efficiency of at least 30%.

16. The method of claim 1, further comprising converting the initial starting genetic material into tagged parent polynucleotides with a conversion efficiency of at least 50%.

17. The method of claim 1, wherein the initial starting genetic material is cell-free nucleic acid.

18. The method of claim 17 wherein analyzing comprises:

(a) identifying a subset of mapped sequence reads that align with a variant of the reference sequence at each mappable base position;

(b) for each mappable base position, calculating a ratio of (1) a number of mapped sequence reads that include a variant as compared to the reference sequence, to (2) a number of total sequence reads for each mappable base position;

(c) normalizing the ratios or frequency of variance for each mappable base position and determining potential rare variant(s) or mutation(s); and

(d) comparing the resulting number for each of the regions with potential rare variant(s) or mutation(s) to similarly derived numbers from a reference sample.

19. The method of claim 18, wherein the prevalence/concentration of each rare variant identified in the subject is reported and quantified simultaneously.

20. The method of claim 18, wherein a confidence score regarding the prevalence/concentrations of rare variants in the subject is reported.

21. The method of claim 18, wherein the normalizing is performed using one or more of hidden Markov, dynamic programming, support vector machine, Bayesian or probabilistic modeling, trellis decoding, Viterbi decoding, expectation maximization, Kalman filtering, or neural network methodologies.

22. The method of claim 18, wherein the genetic profile of a tumor, infection or other tissue abnormality is inferred.

23. The method of claim 17 wherein analyzing comprises:

(a) quantifying or enumerating mapped reads in two or more predefined regions of the reference sequence; and

(b) determining copy number variation in one or more of the predefined regions by;

(1) normalizing number of unique sequence reads in the predefined regions to one another;

(2) comparing the normalized numbers obtained in (1) to normalized numbers obtained from a control sample.

24. The method of claim 1, wherein collapsing comprises:

grouping sequence reads sequenced from amplified progeny polynucleotides into families, each family amplified from the same tagged parent polynucleotide; and

determining a consensus sequence based on sequence reads in a family.

25. The method of claim 1, wherein the initial starting genetic material is tagged with a DNA barcode.

26. The method of claim 1, wherein initial starting genetic material is tagged with polynucleotide barcodes on both ends of a parent polynucleotide by ligation prior to amplification.

27. The method of claim 1, further comprising selectively enriching regions from the subject'"'"'s genome or transcriptome prior to sequencing.

28. The method of claim 1, further comprising non-selectively enriching regions from the subject'"'"'s genome or transcriptome prior to sequencing.

29. The method of claim 1, wherein collapsing uses sequence information from a tag and more than one of:

sequence information at a beginning region of a sequence read, the end region of the sequence read, and the length of the sequence read.

30. The method of claim 1, wherein sequence information from a tag comprises sequence information from barcodes attached at both ends of a double stranded polynucleotide molecule in the starting genetic material.