Multiple tagging of individual long DNA fragments

• US 9,328,382 B2
• Filed: 03/11/2014
• Issued: 05/03/2016
• Est. Priority Date: 03/15/2013
• 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., Alexeev, Andrei
• Primary Examiner(s): Ghafoorian, Reza
• Application Number:
  US14/205,145
• Publication Number:
  US 20140323316A1
• Time in Patent Office: 784 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

This disclosure provides methods and compositions for tagging long fragments of a target nucleic acid for sequencing and analyzing the resulting sequence information in order to reduce errors and perform haplotype phasing, for example.

Claims

1. A method of generating tagged sequence reads comprising:

(a) combining;

(i) a plurality of long fragments of a target nucleic acid, wherein the fragments are from about 5 kilobases to about 750 kilobases in length;

(ii) a population of beads, wherein each bead comprises multiple copies of a single polynucleotide comprising a tag-containing sequence, wherein said tag-containing sequence comprises a tag sequence and a transposon end sequence, and wherein different beads comprise different tag sequences; and

(iii) a transposase,

in a single reaction vessel under conditions in which, for a plurality of beads, the tag sequence associated with an individual bead is transposed into multiple sites of an individual long fragment;

(b) producing a plurality of subfragments of the long fragments; and

,

(c) sequencing subfragments from (b) to produce a plurality of sequence reads including tag sequences.

2. The method of claim 1 wherein said tag-containing sequences comprise a tag sequence and producing tagged subfragments comprises performing an amplification reaction to produce a plurality of amplicons.

3. The method of claim 1 wherein the target nucleic acid is a complex nucleic acid.

4. The method of claim 3 wherein the target nucleic acid is a genome of an organism.

5. The method of claim 1 wherein the length of the tag sequence is from 8 to 12 nucleotides.

6. The method of claim 5 comprising determining a haplotype of the genome.

7. The method of claim 1 wherein the tag-containing sequences comprise a region of degenerate sequence that serves as a tag sequence, a known sequence that is a PCR primer binding site, and a known sequence that is a transposon end sequence.

8. The method of claim 1 further comprising mapping and assembling the sequence reads into genome sequence.

9. The method of claim 1 further comprising assembling the sequence reads to produce an assembled sequence of a long fragment.

10. The method of claim 1 wherein each bead comprises hundreds of copies of the polynucleotide comprising a unique tag-containing sequence.

11. The method of claim 10 wherein each bead comprises thousands of copies of the polynucleotide comprising a unique tag-containing sequence.

12. The method of claim 1 wherein tag sequences of at least some beads are transpose into more than one long fragment.

13. The method of claim 1 wherein for a majority of long fragments tagged at multiple sites, said sites are separated from each other by an average spacing in the range of 100 bp to 5,000 bp.