Processing and Analysis of Complex Nucleic Acid Sequence Data

• US 20130124100A1
• Filed: 04/13/2012
• Published: 05/16/2013
• Est. Priority Date: 06/15/2009
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje, Peters, Brock A., Kermani, Bahram Ghaffarzadeh
• Granted Patent: US 9,524,369 B2
• Time in Patent Office: Days
• US Class Current: 702/20
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2549/10 the purpose being that of reducing false positive or false negative signals
• 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
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

The present invention is directed to logic for analysis of nucleic acid sequence data that employs algorithms that lead to a substantial improvement in sequence accuracy and that can be used to phase sequence variations, e.g., in connection with the use of the long fragment read (LFR) process.

Claims

1. A method of determining a sequence of a complex nucleic acid of one or more organisms, the method comprising:

(a) receiving, at one or more computing devices, a plurality of reads of the complex nucleic acid; and

(b) producing, with the one or more computing devices, an assembled sequence of the complex nucleic acid from the reads, the assembled sequence comprising less than one false single nucleotide variant per megabase at a call rate of 70% or greater.

2. The method of claim 1 further comprising identifying a plurality of sequence variants in said assembled sequence and phasing said plurality of sequence variants to produce a phased sequence.

3. The method of claim 2 comprising phasing at least three of the sequence variants, and identifying as an error a sequence variant that is inconsistent with the phasing of said at least two sequence variants.

4. The method of claim 2 wherein the assembled sequence is a whole genome sequence, the method comprising phasing at least 70 percent of the sequence variants.

5. The method of claim 2 wherein the assembled sequence is a whole genome sequence, the method comprising phasing at least 80 percent of the sequence variants.

6. The method of claim 2 wherein the assembled sequence is a whole genome sequence, the method comprising phasing at least 85 percent of the sequence variants.

7. The method of claim 2 wherein the assembled sequence is a whole genome sequence, the method comprising phasing at least 90 percent of the sequence variants.

8. The method of claim 2 wherein the assembled sequence is a whole genome sequence, the method comprising phasing at least 95 percent of the sequence variants.

9. The method of claim 1 wherein said step of receiving said plurality of reads of the complex nucleic acid consists of receiving a plurality of reads from each of a plurality of aliquots, each aliquot comprising one or more fragments of the complex nucleic acid.

10. The method of claim 9 comprising calling a base at a position of said assembled sequence on the basis of preliminary base calls for the position from two or more aliquots.

11. The method of claim 9 comprising identifying the base call as true if it is present 3 or more times in reads from two or more aliquots.

12. The method of claim 9 wherein an aliquot-specific tag is attached to each of the fragments, the method further comprising determining the aliquot from which the reads originate by identifying the aliquot-specific tag.

13. The method of claim 12 wherein the aliquot-specific tag comprises an error-correction code, and each read comprises tag sequence data and fragment sequence data, wherein the tag sequence data is either correct tag sequence data or incorrect tag sequence data that comprises one or more errors;

the method further comprising;

(c) using the error-correction code to correct the incorrect tag sequence data, thereby producing corrected tag sequence data and tag sequence data that cannot be corrected;

(d) using reads comprising the correct tag sequence data and the corrected tag sequence data in a first computer process that requires tag sequence data and that produces a first output; and

(e) using reads comprising the tag sequence data that cannot be corrected in a second computer process that does not require tag sequence data and that produces a second output.

14. The method of claim 13 wherein said first computer process is selected from the list comprising sample multiplexing, library multiplexing, phasing, and an error correction process that employs tag sequence data.

15. The method of claim 13 wherein the second computer process comprises mapping, assembly, and pool-based statistics.

16. The method of claim 13 wherein the error-correction code is a Reed-Solomon code.

17. The method of claim 1 wherein the method further comprises:

(c) providing a first phased sequence of a region of the complex nucleic acid, the region comprising a short tandem repeat;

(d) comparing reads of the first phased sequence of the region with reads of a second phased sequence of the region; and

(e) based on the comparison, identifying an expansion of the short tandem repeat in one of the first phased sequence or the second phased sequence.

18. The method of claim 1 further comprising obtaining genotype data from at least one parent of the organism and producing an assembled sequence of the complex nucleic acid from the reads and the genotype data of the at least one parent.

19. The method of claim 1 further comprising adding population genotype data and producing an assembled sequence of the complex nucleic acid from the reads and the population genotype data.

20. The method of claim 1 further comprising:

(c) aligning a plurality of the reads for a first region of the complex nucleic acid, thereby creating an overlap between the aligned reads;

(d) identifying N candidate hets within the overlap, where N is an integer greater than two;

(e) clustering a space of 2^N to 4^N possibilities for the N candidate hets or a selected subspace of said space, thereby creating a plurality of clusters;

(f) identifying two clusters with the highest density, each identified cluster comprising a substantially noise-free center; and

(g) repeating steps (a)-(d) for one or more additional regions of the complex nucleic acid.

21. The method of claim 1 wherein the assembled sequence comprises fewer than 0.8 false single nucleotide variants per megabase.

22. The method of claim 1 wherein the assembled sequence comprises fewer than 0.6 false single nucleotide variants per megabase.

23. The method of claim 1 wherein the assembled sequence comprises fewer than 0.4 false single nucleotide variants per megabase.

24. The method of claim 1 wherein the assembled sequence comprises fewer than 0.2 false single nucleotide variants per megabase.

25. The method of claim 1 wherein the assembled sequence comprises fewer than 0.1 false single nucleotide variants per megabase.

26. The method of claim 1 wherein the assembled sequence has a call rate of at least 80 percent of the complex nucleic acid.

27. The method of claim 1 wherein the assembled sequence has a call rate of at least 85 percent.

28. The method of claim 1 wherein the assembled sequence has a call rate of at least 90 percent.

29. The method of claim 1 further comprising:

(a) providing an amount of the complex nucleic acid, and (b) sequencing said amount of the complex nucleic acid to produce said plurality of reads.

30. The method of claim 1 wherein the complex nucleic acid is selected from the group consisting of a genome, an exome, a transcriptome, a methylome, a mixture of genomes of different organisms, a mixture of genomes of different cell types of an organism, and subsets thereof.

31. The method of claim 1 wherein the organism is a mammal.

32. The method of claim 1 wherein the organism is a human.

33. One or more computer-readable non-transitory storage media storing an assembled human genome sequence produced by the method of claim 1.

34. A computer-readable non-transitory storage medium storing instructions which, when executed by one or more computing devices, cause the one or more computing devices to perform the method of claim 1.

36. The method of claim 34 wherein the assembled sequence of the human genome comprises a genome call rate of 70% and an exome call rate of 70% or greater.

35. A method of determining a sequence of a human genome, the method comprising:

(a) receiving at one or more computing devices, a plurality of reads of the genome; and

(b) producing, with the one or more computing devices, an assembled sequence of the genome from the reads, the assembled sequence comprising less than 600 false single nucleotide variants per gigabase at a genome call rate of 70% or greater.

37. A computer-readable non-transitory storage medium storing instructions which, when executed by one or more computing devices, cause the one or more computing devices to perform the method of claim 35.

38. A method of determining a sequence of a human genome, the method comprising:

(a) receiving, at one or more computing devices, a plurality of reads from each of a plurality of aliquots, each aliquot comprising a fragment of the human genome; and

(b) producing, with the one or more computing devices, a phased, assembled sequence of the genome from the reads, the assembled sequence comprising less than 1000 false single nucleotide variants per gigabase at a genome call rate of 70% or greater.

39. A computer-readable non-transitory storage medium storing instructions which, when executed by one or more computing devices, cause the one or more computing devices to perform the method of claim 38.