METHODS FOR OBTAINING A SEQUENCE

• US 20130079231A1
• Filed: 09/10/2012
• Published: 03/28/2013
• Est. Priority Date: 09/09/2011
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Quake, Stephen R., Voskoboynik, Ayelet, Kertesz, Michael, Pushkarev, Dmitry
• Granted Patent: US 9,249,460 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 2527/146 Concentration of target or template
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention generally relates to methods for obtaining a sequence, such as a consensus sequence or a haplotype sequence. In certain embodiments, methods of the invention involve determining an amount of amplifiable nucleic acid present in a sample, partitioning the nucleic acid based upon results of the determining step such that each partitioned portion includes, on average, a subset of unique sequences, sequencing the nucleic acid to obtain sequence reads, and assembling a consensus sequence from the reads.

Claims

1. A method for obtaining a sequence, the method comprising:

determining an amount of amplifiable nucleic acid present in a sample;

partitioning the nucleic acid based upon results of the determining step such that each partitioned portion comprises, on average, a subset of unique sequences;

sequencing the nucleic acid to obtain sequence reads; and

obtaining a sequence from the reads.

2. The method according to claim 1, wherein prior to the determining step, the method further comprises fragmenting the nucleic acid.

3. The method according to any one of claims 1 or 2, further comprising attaching a set of unique marker identifier to ends of each fragment.

4. The method according to claim 3, wherein the unique marker identifiers comprise bar code sequences that are part of adapter sequences.

5. The method according to claim 4, wherein the bar code sequences are the same or different.

6. The method according to claim 4, wherein the adapter sequences are amplification adaptors.

7. The method according to any one of claim 3, further comprising attaching labels to the nucleic acids in each partitioned portion.

8. The method according to claim 7, wherein prior to the attaching labels step, the method further comprises amplifying the nucleic acids in each partitioned portion.

9. The method according to claim 8, wherein after the amplifying step, the method further comprises fragmenting the amplified nucleic acids in each partitioned portion.

11. The method according to claim 1, wherein partitioning comprises dispensing the sample into different wells of a microwell plate.

12. The method according to claim 1, wherein partitioning comprises segmenting the sample into droplets.

13. The method according to claim 1, wherein the sequence is a consensus sequence or a haplotype sequence.

14. A method for obtaining a sequence, the method comprising:

obtaining nucleic acid;

fragmenting the nucleic acid;

determining an amount of amplifiable nucleic acid present in a sample;

partitioning the fragmented nucleic acids based upon results of the determining step;

amplifying the partitioned nucleic acids;

attaching bar codes to the amplified nucleic acid;

sequencing the nucleic acid to obtain bar coded sequence reads; and

obtaining a sequence from the reads.

15. The method according to claim 14, wherein each partitioned portion comprises, on average, a unique subset of nucleic acids.

16. The method according to claim 14, further comprising attaching marker identifiers to ends of the fragments.

17. The method according to claim 14, wherein partitioning comprises dispensing the sample into different wells of a microwell plate.

18. The method according to claim 14, wherein partitioning comprises segmenting the sample into droplets.

19. The method according to claim 14, wherein the sequence is a consensus sequence or a haplotype sequence.