MULTIPLE TAGGING OF INDIVIDUAL LONG DNA FRAGMENTS

• US 20170022554A1
• Filed: 04/22/2016
• Published: 01/26/2017
• 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
• Granted Patent: US 10,023,910 B2
• Time in Patent Office: 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 sequencing a target nucleic acid comprising:

(a) combining in a single reaction vessel (i) a plurality of long fragments of the target nucleic acid, and (ii) a population of polynucleotides, wherein each polynucleotide comprises a tag and a majority of the polynucleotides comprise a different tag;

(b) introducing into a majority of the long fragments tag-containing sequences from said population of polynucleotides to produced tagged long fragments, wherein each of the tagged long fragments comprises a plurality of the tag-containing sequences at a selected average spacing, and each tag-containing sequence comprises a tag;

(c) producing a plurality of subfragments from each tagged long fragment, wherein each subfragment comprises one or more tags;

(d) sequencing the subfragments to produce a plurality of sequence reads;

(e) assign a majority of the sequence read to corresponding long fragments; and

assembling the sequence reads to produce an assembled sequence of the target

2. The method of claim 1 wherein producing the subfragments comprises performing an amplification reaction to produce a plurality of amplicons from each long fragment.

3. The method of claim 2 wherein each amplicon comprises a tag from each of the adjacent introduced sequences and a region of the long fragment between the adjacent introduced sequences.

4. The method of claim 1 comprising combining the long fragments with an excess of the population of tag-containing sequences.

5. The method of claim 1 comprising combining the long fragments with the tag-containing solution under conditions that are suitable for introduction of a single tag-containing sequence into a majority of the long fragments.

6. The method of claim 1 comprising combining the long fragments with the tag-containing solution under conditions that are suitable for introduction of a different tag-containing sequences into a majority of the long fragments.

7. The method of claim 1 wherein the population of tag-containing sequences comprises a population of beads, wherein each bead comprises multiple copies of a single tag-containing sequence.

8. The method of claim 1 wherein the population of tag-containing sequences comprises a population of concatemers, each concatemer comprising multiple copies of a single tag-containing sequence.

9. The method of claim 1 wherein the tag-containing sequences comprise transposon ends, the method comprising combining the long fragments and the tag-containing sequences under conditions that are suitable for transposition of the tag-containing sequences into each of the long fragments.

10. The method of claim 1 wherein the tag-containing sequences comprise a hairpin sequence.

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

12. The method of claim 1 wherein the target nucleic acid is a genome of an organism.

13. The method of claim 12 comprising determining a haplotype of the genome