Sequence assembly
The invention relates to assembly of sequence reads. The invention provides a method for identifying a mutation in a nucleic acid involving sequencing nucleic acid to generate a plurality of sequence reads. Reads are assembled to form a contig, which is aligned to a reference. Individual reads are aligned to the contig. Mutations are identified based on the alignments to the reference and to the contig.
1. A method comprising:
receiving, onto a memory, a plurality of sequence reads from nucleic acid from a sample organism;
assembling the plurality of sequence reads into an assembly that represents a contiguous region of the nucleic acid;
aligning the assembly to the reference genome to generate an assembly-reference alignment;
identifying a plurality of variants in the assembly relative to the reference genome;
aligning, by pairwise alignment, each of the plurality of sequence reads to the assembly to generate a read-assembly alignment; and
assigning a variant state for each of the plurality of variants relative to the reference genome based on the assembly-reference alignment and the read-assembly alignment to determine genotype information for the sample organism.
2. The method of claim 1 , wherein assembling the plurality of sequence reads comprises mapping the plurality of sequence reads to the reference genome using Burrows-Wheeler Aligner (BWA) to produce mapped reads, and assembling the mapped reads via a de-Bruijn graph-based method into the assembly.
3. The method of claim 1 , wherein the variant state is selected from a mutation, an insertion, or a deletion.
4. The method of claim 1 , wherein aligning each of the plurality of sequence reads to the assembly generates a likelihood score for each of the plurality of sequence reads.
5. The method of claim 4 , wherein the likelihood score assigns a variant state to each of the plurality of sequence reads.
6. The method of claim 1 , wherein aligning the assembly to the reference genome comprises applying at least a Smith-Waterman algorithm to the assembly and the reference genome.
7. The method of claim 1 , wherein the assembly-reference alignment and locations of the plurality of variants are described in a Concise Idiosyncratic Gapped Alignment Report (CIGAR) string.
8. The method of claim 1 , further comprising genotyping the sample organism based on the variant state of one or more of the plurality of variants.
9. The method of claim 1 , wherein the variant state for each of the plurality of variants are output in a Variant Call Format (VCF) file.
10. A method for genotyping nucleic acids, the method comprising:
obtaining a sample comprising nucleic acid;
sequencing the nucleic acid to generate a plurality of sequence reads; and
detecting a variant in the plurality of sequence reads relative to a reference genome, the detecting comprising the steps of:
assembling a contig from the plurality of sequence reads;
aligning the contig to the reference genome to obtain a contig-to-reference alignment, the contig-to-reference alignment comprising a position of the contig relative to the reference genome and a description of one or more differences in the aligned contig relative to the reference genome;
aligning each of the plurality of sequence reads to the aligned contig to obtain a plurality of read-to-contig alignments, the plurality of read-to-contig alignments comprising, for each of the plurality of sequence reads, a position of the sequence read relative to the aligned contig and a description of one or more differences in the aligned sequence read relative to the aligned contig;
aligning the contig-to-reference alignment to the plurality of read-to-contig alignments to obtain a plurality of read-to-reference alignments, the plurality of read-to-reference alignments comprising, for one or more of the plurality of sequence reads, a position of the one or more of the plurality of sequence reads relative to the reference genome and a description of a difference in the one or more plurality of sequence reads relative to the reference genome; and
outputting the difference as the detected variant.
11. The method of claim 10 , further comprising inputting the plurality of sequence reads into a computer system comprising a processor and a main memory coupled to a non-transitory memory, wherein the computer system performs the detecting step.
12. The method of claim 11 , wherein the reference genome is stored in the main memory.
13. The method of claim 12 , wherein the aligning the contig to the reference genome comprises applying a Burrows-Wheeler Aligner (BWA)-long algorithm.
14. The method of claim 13 , wherein the BWA-long algorithm returns a Binary Alignment Map (BAM) file comprising the contig-to-reference alignment.
15. The method of claim 14 , wherein the description of the one or more differences in the aligned contig relative to the reference genome are described in a Concise Idiosyncratic Gapped Alignment Report (CIGAR) string.
16. The method of claim 11 , wherein the step of aligning each of the plurality of sequence reads to the aligned contig comprises applying a BWA-short algorithm.
17. The method of claim 16 , wherein the BWA-short algorithm returns a BAM file comprising the plurality of read-to-contig alignments.
18. The method of claim 17 , wherein the description of the one or more differences in the aligned sequence read relative to the aligned contig is described in a read-to-contig CIGAR string.
19. The method of claim 18 , wherein the read-to-contig CIGAR string is adjusted to include the plurality of read-to-reference alignments to produce a read-to-reference CIGAR string.
20. The method of claim 19 , wherein the read-to-reference CIGAR string identifies a variant relative to the reference genome.