Nucleic acid library generation methods and compositions

• US 10,647,981 B1
• Filed: 09/08/2016
• Issued: 05/12/2020
• Est. Priority Date: 09/08/2015
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Luckey, John A.
• Primary Examiner(s): Vivlemore, Tracy
• Assistant Examiner(s): Kaup, Sahana S
• Application Number:
  US15/259,899
• Time in Patent Office: 1,342 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

Methods of forming a library of polynucleotide templates for DNA sequencing are disclosed. The methods include distributing a nucleic acid sample into different droplets in a set of emulsion droplets. The droplets into which the polynucleotide species are distributed include primers which binds to the polynucleotide species and are extended by a polymerase contained within the droplet. A set of extended primers are created simultaneously in a large number of droplets. An enriched set of extended primers are distributed into a second set of emulsion droplets containing a second primer which is extended by a polymerase contained within the second set of droplets.

Claims

1. A method of forming a library of polynucleotide templates for sequencing, the method comprising the steps of:

distributing a nucleic acid sample comprising a plurality of different polynucleotide species into a first set of emulsion droplets, wherein a plurality of droplets comprise a single polynucleotide species and a first primer comprising;

(1) a region complementary to the polynucleotide species;

(2) a region for binding to a first universal primer sequence; and

(3) a purification binding moiety;

extending the first primer, whereby a set of extended first primers are created;

breaking the first emulsion droplets;

creating an enriched set of extended first primers;

distributing members of the enriched set of extended first primers into a second set of emulsion droplets, wherein a plurality of droplets comprise a single first extended primer species and a second primer comprising;

(1) a region complementary to the extended primer; and

(2) a region for binding to a second universal primer sequence; and

extending the second primer in the droplets, whereby a library of polynucleotide templates comprising the first and second universal primer binding sequences are created.

2. The method of claim 1, further comprising the steps of:

binding the extended polynucleotide template to a solid support though the purification binding moiety;

washing the bound polynucleotide constructs to remove unbound polynucleotides; and

releasing a strand of the bound polynucleotide templates, whereby a set or released polynucleotide templates is produced.

3. The method of claim 2, further comprising:

distributing the set of released polynucleotide templates into third set of emulsion droplets, wherein the droplets comprise;

(1) a first adapter primer comprising a region for binding to the region for binding to the first universal primer sequence; and

(2) a second adapter primer comprising a region for binding to the region for binding to the second universal primer sequence.

4. The method of claim 3, wherein the first adapter primer or the second adapter primer comprises a sample index sequence.

5. The method of claim 1, wherein the first primer comprises a unique identifier sequence.

6. The method of claim 1, wherein the region complementary to the polynucleotide species on the first primer is targeted to specific sequence.

7. The method of claim 6, wherein the region complementary to the extended first primer on the second primer is targeted to specific sequence.

8. The method of claim 1, wherein the enriched set of extended first primers is created by a process comprising the step of contacting the set of extended primers with solid phase reversible immobilization beads.

9. The method of claim 8, wherein the solid phase reversible immobilization beads are AMPURE BEADS™

.

10. The method of claim 1, comprising the step of sequencing a plurality of the library of polynucleotide templates.

11. The method of claim 1, wherein the region complementary to the extended first primer on the second primer is random sequence, and the step of extending the second primer produces a plurality extended second primers having different random sequences, and the extending is catalyzed by an enzyme have strand displacement activity or 5′

to 3′

exonuclease activity.

12. The method of claim 1, wherein the nucleic acid sample has been enriched for specific target prior to distributing polynucleotide species in the first set of emulsion droplets.

13. The method of claim 1, wherein the nucleic acid sample is a cDNA library.