Multiple tagging of individual long DNA fragments

Patent metadata

Field	Value
Publication number	US-9328382-B2
Application number	US-201414205145-A
Country	US
Kind code	B2
Filing date	Mar 11, 2014
Priority date	Mar 15, 2013
Publication date	May 3, 2016
Grant date	May 3, 2016

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.

First claim

The invention claimed is:

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.

Assignees

• Complete Genomics Inc

Inventors

• Drmanac Radoje
• Peters Brock A
• Alexeev Andrei

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2525/204

  specific length of the oligonucleotides · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title