Methods for obtaining a sequence

• US 9,249,460 B2
• Filed: 09/10/2012
• Issued: 02/02/2016
• 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: Pushkarev, Dmitry, Quake, Stephen R., Voskoboynik, Ayelet, Kertesz, Michael
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Zhang, Kaijiang
• Application Number:
  US13/608,778
• Publication Number:
  US 20130079231A1
• Time in Patent Office: 1,240 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 506/7, 506/2
• US Class Current: 1/1
• 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:

ligating adapters to a plurality of nucleic acid fragments in a sample, each nucleic acid fragment having a length between 5 kilobases and 40 kilobases;

quantifying the amount of nucleic acid fragments present in the sample to which adapters have been ligated on each end, thereby quantifying the amount of amplifiable nucleic acid fragments;

determining a dilution factor based on the quantified amount of amplifiable nucleic acid fragments;

partitioning the sample based on the dilution factor such that each partitioned portion comprises, on average, at least two amplifiable nucleic acid fragments that are a subset of unique sequences;

performing a polymerase chain reaction on the partitioned nucleic acid fragments;

sequencing the nucleic acid fragments to obtain sequence reads; and

obtaining a sequence from the reads.

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

3. The method according to claim 2, wherein the adapters comprise unique marker identifiers.

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 in the adapters attached to both ends of a fragment are the same or different.

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

7. The method according to claim 3, further comprising attaching labels to the nucleic acid fragments in each partitioned portion.

8. The method according to claim 7, wherein the attaching labels step is performed after the polymerase chain reaction.

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

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

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

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

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

obtaining nucleic acid;

fragmenting the nucleic acid, thereby generating nucleic acid fragments that have a length of 5 kilobases to 40 kilobases;

ligating adapters to a portion of the nucleic acid fragments, wherein the portion of nucleic acid fragments with adapters ligated at each end are amplifiable nucleic acid fragments;

quantifying an amount of the amplifiable nucleic acid fragments present in the sample;

determining a dilution factor based on the quantified amount of amplifiable nucleic acid fragments;

partitioning the nucleic acid fragments based on the dilution factor, wherein each partitioned portion comprises, on average, at least two amplifiable nucleic acid fragments that are a subset of unique sequences;

amplifying the partitioned nucleic acid fragments by performing a polymerase chain reaction;

attaching bar codes to the amplified nucleic acid fragments;

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

obtaining a sequence from the reads.

14. The method according to claim 13, wherein each adapter comprises a marker identifiers.

15. The method according to claim 13, wherein partitioning comprises dispensing the sample into different wells of a microwell plate.

16. The method according to claim 13, wherein partitioning comprises segmenting the sample into droplets.

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