Targeted Sequencing and UID Filtering

Patent metadata

Field	Value
Publication number	US-2016201124-A1
Application number	US-201514910237-A
Country	US
Kind code	A1
Filing date	Feb 11, 2015
Priority date	Feb 11, 2014
Publication date	Jul 14, 2016
Grant date	—

Abstract

Provided herein are methods, compositions, and kits for targeted sequencing of polynucleotides with high accuracy and low amplification and sequencing errors.

First claim

1 . A method of generating a library of polynucleotides comprising:
(a) generating a first complement sequence (CS) of a target polynucleotide from a sample using a first primer, the first primer comprising a target specific sequence; (b) attaching to the first CS an adaptor comprising a first primer binding sequence (PBS) or portion thereof, thereby forming a modified compliment sequence (MCS); (c) extending a second primer hybridized to the MCS, thereby forming a second CS, wherein the second primer comprises: (i) a target specific region, and (ii) a second PBS; and (d) amplifying the second CS using primers that hybridize to the first PBS and second PBS respectively, wherein the first or the second primer comprises a unique identification (UID) sequence.

2 . A method of generating a library of polynucleotides comprising:
(a) extending target specific first primer hybridized to a target polynucleotide to form a first CS; (b) attaching an adaptor to the first CS to form an MCS; (c) extending a second primer hybridized to the MCS to form a second CS; and (d) amplifying the second CS; wherein (a) or (c) do not comprise exponential amplification, and wherein the first or the second primer comprises a UID.

3 . A method of generating a library of polynucleotides comprising:
(a) generating a first CS, or modified form thereof (MCS), from a target polynucleotide; (b) generating a second CS from a polynucleotide comprising the sequence of the first CS; wherein the second CS is generated by a non-exponential amplification reaction; and (c) amplifying the second CS; wherein the first CS or the second CS comprises a UID.

4 . A method of accurately determining the sequence of a target polynucleotide comprising:
(a) generating a second CS from a first CS, or modified form thereof (MCS), generated from a target polynucleotide; wherein the first CS, second CS, or MCS comprises a UID, and wherein the first and second CSs are each individually generated by (i) a primer extension reaction, or (ii) a linear amplification reaction; (b) amplifying the second CS; (c) sequencing at least one of the amplified second CSs; (d) aligning at least two sequences containing the same UID from (c); and (e) determining a consensus sequence based on (d), wherein the consensus sequence accurately represents the target polynucleotide sequence.

5 . The method of claim 4 , wherein the first primer comprises a universal ligation sequence (ULS).

6 . The method of claim 4 , wherein the second primer further comprises a universal priming sequence (UPS).

7 . The method of claim 4 , wherein the adapter further comprises a sample barcode (SBC) sequence.

8 . The method of claim 4 , wherein the MCS further comprises an affinity molecule or capture sequence.

9 . The method of claim 4 , wherein the UID comprises the sequence NNNNNNNNNNNNNNN (SEQ ID NO: 1), wherein N is any nucleic acid residue.

10 . The method of claim 4 , wherein the UID comprises the sequence NNNNNWNNNNNWNNNNN (SEQ ID NO: 2), wherein N is any nucleic acid residue and W is adenine or thymine.

11 . The method of claim 4 , wherein the attaching comprises ligating.

12 . The method of claim 4 , wherein the attaching comprises amplification.

13 . A method of accurately determining the sequence of a target polynucleotide comprising:
(a) generating a second CS from a first CS, or modified form thereof (MCS), generated from a target polynucleotide; wherein the first and second CSs are each individually generated by (i) a primer extension reaction, or (ii) a linear amplification reaction; (b) amplifying the second CS; (c) sequencing at least one of the amplified second CSs; (d) aligning at least two sequences from (c) comprising at least 10% sequence identity; and (e) determining a consensus sequence based on (d), wherein the consensus sequence accurately represents the target polynucleotide sequence.

14 . A method of accurately determining the sequence of a target polynucleotide comprising:
(a) generating a library according to claim 12 , (b) sequencing at least a portion of said library (c) aligning at least two sequences from (b) comprising at least 10% sequence identity; and determining a consensus sequence based on (c), wherein the consensus sequence accurately represents the target polynucleotide sequence.

Assignees

• Roche Molecular System Inc
• Roche Molecular Systems Inc

Inventors

• Donahue William
• Vigneault Francois

Classifications

• C12N15/1096 Primary

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• C12Q1/6806

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