Sequence assembly

• US 10,604,799 B2
• Filed: 04/11/2014
• Issued: 03/31/2020
• Est. Priority Date: 04/04/2012
• Status: Active Grant

• Current Assignee: Invitae Corporation
• Original Assignee: Molecular Loop Biosolutions, LLC
• Inventors: Porreca, Gregory, Kennedy, Caleb
• Primary Examiner(s): Brusca, John S
• Application Number:
  US14/250,891
• Publication Number:
  US 20140255931A1
• Time in Patent Office: 2,181 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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 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.

Claims

1. A method for assembling sequence reads, the method comprising:

obtaining a sample comprising template nucleic acid;

sequencing the template nucleic acid to generate a plurality of sequence reads;

inputting a reference genome and said plurality of sequence reads into

a computer system comprising a processor coupled to a non-transitory memory to perform the steps of;

assembling a contig from at least some of the plurality of sequence reads;

identifying a plurality of contig;

reference descriptions of mutations by aligning the contig to said reference genome;

identifying a plurality of read;

contig descriptions by aligning each of the plurality of sequence reads to the contig; and

combining the contig;

reference descriptions with the read;

contig descriptions to produce read;

reference descriptions to map positional information of mutations found in the individual reads relative to the reference.

2. The method of claim 1, further comprising attaching barcode sequences to the template nucleic acid.

3. The method of claim 2, further comprising assigning the reads to subsets based on the barcode sequences.

4. The method of claim 3, wherein each subset is used in creating a unique contig for that subset.

5. The method of claim 1, wherein sequencing the template nucleic acid comprises fragmenting the template nucleic acid, attaching the fragments to a surface of channels in a flow cell, and amplifying the attached fragments to create clusters, each cluster comprising a plurality of copies of the same template in one of the channels of the flow cell.

6. The method of claim 1, wherein creating the contig comprises overlap consensus assembly.

7. The method of claim 1, wherein creating the contig comprises searching a prefix tree for overlap between the reads.

8. The method of claim 1, further comprising identifying a mutation based on the alignments to the contig and the reference sequence.

9. The method of claim 8, wherein a plurality of mutations are identified.

10. The method of claim 9, wherein a first mutation is within about 100 nucleotides of a second mutation.

11. The method of claim 10, wherein the first mutation is a substitution and the second mutation is a deletion.

12. The method of claim 8, wherein the mutation is a deletion at an end of a sequence read.

13. The method of claim 1, wherein sequencing is sequencing-by-synthesis.

14. The method of claim 13, wherein sequencing-by-synthesis is single molecule sequencing-by-synthesis.

15. The method of claim 1, wherein aligning the contig to a reference sequence is performed using a first set of alignment parameters and aligning each of the plurality of sequence reads to the contig is performed using a second set of alignment parameters.

16. The method of claim 15, wherein the first set of alignment parameters includes a first gap penalty and a first substitution penalty;

the second set of alignment parameters includes a second gap penalty and a second substitution penalty; and

the first gap penalty>

the second gap penalty.