Polony sequencing methods

• US 9,982,296 B2
• Filed: 09/30/2015
• Issued: 05/29/2018
• Est. Priority Date: 10/09/2009
• Status: Active Grant

• Current Assignee: UNM Rainforest Innovations (f/k/a STC.UNM) (The University of New Mexico)
• Original Assignee: UNM Rainforest Innovations (f/k/a STC.UNM) (The University of New Mexico)
• Inventors: Edwards, Jeremy
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/870,513
• Publication Number:
  US 20160194700A1
• Time in Patent Office: 972 Days
• Field of Search: None
• 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 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

We describe ultra-high throughput polony genome sequencing that can permit, for example, generating raw data to re-sequencing the human genome in about one week (including library prep and sequencing) at a reasonable cost. The methods described herein include one or more of the following: (1) increasing polony sequencing read length, (2) improving library construction and emulsions protocols, (3) increasing bead density and/or moving to alternative clonal amplication strategies (other than emulsion PCR or ePCR), (4) extending software capabilities to allow SNP calls from our new sequencing raw data, (5) Dual Primer Emulsion PCR, and (6) diagnostic method exploiting one or more of the foregoing.

Claims

1. A method for obtaining the sequence of a polynucleotide comprising an unknown sequence, the method comprising:

providing a polynucleotide library comprising a plurality of polynucleotide fragments comprising unknown polynucleotide sequences;

providing a substrate comprising a set of polynucleotide fragments having known nucleotide sequences from a reference genome;

sequencing the ends of the library of polynucleotide fragments, thereby creating partially-sequenced fragments;

comparing the sequenced ends of the partially-sequenced fragments to the reference genome;

sequencing the rest of the partially-sequenced fragments, wherein sequencing the partially-sequenced fragments comprises;

contacting at least a portion of the partially-sequenced fragments with the substrate under conditions suitable to allow the partially-sequenced fragments to hybridize with a complementary substrate polynucleotide fragment; and

detecting the hybridizations; and

obtaining the sequence of the entire partially-sequenced fragments from the known polynucleotide sequence of reference genome to which the partially-sequenced fragments hybridize.

2. The method of claim 1 wherein sequencing the partially-sequenced fragments comprises assigning to each partially-sequenced fragment a nucleic acid sequence that is the complement of the substrate polynucleotide fragment to which it hybridized.

3. The method of claim 1 wherein sequencing the ends of the library of polynucleotide fragments comprises sequencing by chemical synthesis.