Compositions and methods for identification of a duplicate sequencing read

• US 10,570,448 B2
• Filed: 01/13/2017
• Issued: 02/25/2020
• Est. Priority Date: 11/13/2013
• Status: Active Grant

• Current Assignee: Tecan Genomics
• Original Assignee: Tecan Genomics
• Inventors: Amorese, Douglas, Scolnick, Jonathan, Schroeder, Ben
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US15/406,002
• Publication Number:
  US 20170240963A1
• Time in Patent Office: 1,138 Days
• 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 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The present invention provides methods, compositions and kits for detecting duplicate sequencing reads. In some embodiments, the duplicate sequencing reads are removed.

Claims

1. A kit comprising:

in a suitable container;

a plurality of synthetic forward adaptors, wherein each forward adaptor is at least partially double-stranded and comprises;

(i) an indexing primer binding site;

(ii) an indexing site;

(iii) an identifier site consisting of between 3 and 8 nucleotides, wherein each identifier site has a sequence that differs from the every other identifier site sequence in the kit at at least three nucleotide positions; and

(iv) a target sequencing primer binding site comprising SEQ ID NO;

3;

wherein each indexing site is unique amongst a subset of the adaptors and is an index for multiple polynucleotides, thus allowing for samples to be pooled together for a multiplexed sequencing run; and

wherein the sequence of the identifier site is variable in sequence content in the plurality of adaptors and is used to identify duplicate sequence reads in the multiplexed sequencing run.

2. The kit of claim 1 further comprising, in one or more further suitable containers one or more oligonucleotide primers, and reagents for amplification.

3. The kit of claim 1, wherein the adaptors are in an aqueous solution.

4. The kit of claim 1, wherein the adaptors are lyophilized.

5. The kit of claim 1 further comprising, in one or more additional suitable containers, one or more of the following reagents:

reverse adapter, and

one or more buffers suitable for one or more of the elements contained in said kit.

6. The kit of claim 1, wherein the adaptors comprise from 5′

to 3′

;

a) the indexing primer binding site;

b) the indexing site;

c) the identifier site; and

d) the target sequence primer binding site.

7. The kit of claim 1, wherein the adaptors comprise from 5′

to 3′

;

a) the indexing primer binding site;

b) the indexing site;

c) the target sequence primer binding site; and

d) the identifier site.

8. The kit of claim 1, wherein the indexing site is about 6 nucleotides in length.

9. The kit of claim 1, wherein the identifier site is 6 nucleotides in length.

10. The kit of claim 1, wherein the identifier site is 8 nucleotides in length.

11. The kit of claim 1, wherein the indexing primer binding site is a universal indexing primer binding site.

12. The kit of claim 1, wherein the identifier site is 5′

to the indexing site.

13. The kit of claim 1, wherein the indexing primer binding site in each of the plurality of synthetic double-stranded adaptors comprises SEQ ID NO:

2.

14. The kit of claim 13, further comprising, in one or more additional suitable containers:

DNA ligase, DNA-dependent DNA polymerase, or a RNA-dependent DNA polymerase.