Long-Range Barcode Labeling-Sequencing

• US 20130130919A1
• Filed: 10/16/2012
• Published: 05/23/2013
• Est. Priority Date: 10/18/2011
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Zhang, Tao, Pennacchio, Len A., Froula, Jeff L., CHEN, Feng, Singh, Kanwar K., Eng, Kevin S.
• Granted Patent: US 9,469,874 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme
• 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 2563/179 the label being a nucleic acid

Abstract

Methods for sequencing single large DNA molecules by clonal multiple displacement amplification using barcoded primers. Sequences are binned based on barcode sequences and sequenced using a microdroplet-based method for sequencing large polynucleotide templates to enable assembly of haplotype-resolved complex genomes and metagenomes.

Claims

1. A microdroplet-based method for sequencing large polynucleotide templates, comprising

a) providing a template droplet library, wherein each droplet in said library having a single template polynucleotide, polymerase enzyme and deoxynucleotides in a microdroplet, wherein the polynucleotide is 10 kb or larger;

b) providing a primer droplet library, wherein each droplet in said library having a set of barcoded primers having a sequence comprising sequence shared by all primers at the 5′

-end, followed by a primer specific base barcode sequence in the middle, a random sequence at the 3′

-end and a label;

c) merging each template droplet with a primer droplet to form a merged droplet;

d) amplifying the large polynucleotide templates by multiple displacement amplification (MDA) in each of said merged droplets to form amplified large polynucleotides, wherein the 5′

-end of each amplified large polynucleotide is labeled by a bar-coded primer;

e) breaking emulsion of said merged droplets to recover the amplified large polynucleotides;

f) processing said amplified large polynucleotides by shearing, repairing ends and ligating linkers for sequencing of each amplified large polynucleotide;

g) sequencing of said amplified large polynucleotides labeled by said bar-coded primers.

2. The microdroplet-based method of claim 1, wherein said polynucleotide is 10 to 40 kb.

3. The microdroplet-based method of claim 1, wherein said polynucleotide is linear or circular and the polymerase is phi29 or other strand displacement DNA polymerase.

4. The microdroplet-based method of claim 1, wherein the primer specific base barcode sequence is 16 base pairs long and the sequence shared by all primers and the random sequence at the 3′

end are both 6 base pairs.

5. The microdroplet-based method of claim 1, wherein the 3′

end of the random sequence contains two nucleotides labeled with thiophosphate modification.

6. The microdroplet-based method of claim 1, further comprising a step following the processing step (f) of enriching the barcode labeled amplified large polynucleotide template.

7. The microdroplet-based method of claim 1, further comprising following the amplification step (d) a step of capturing bar-coded amplified large polynucleotide templates using streptavidin attached to a surface, wherein the label of said barcoded primers is biotin.

8. The microdroplet-based method of claim 1, wherein the providing step (b) may occur in two stages, wherein the first stage, random hexamers are used to amplify the large polynucleotide templates in the merged droplets, and in the second stage, the template droplets are merged with primer droplets containing barcoded primers for further amplification and introducing barcoded sequences into the amplified products.