# US 10,865,444 B2 · Amplicon preparation and sequencing on solid supports

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- Number: US 10,865,444 B2
- Document ID: US10865444B2
- Status: Granted
- Kind: B2
- Application number: 15/935,897
- Filed: 2018-03-26
- Published: 2018-10-11
- Granted: 2020-12-15
- Earliest claimed: 2014-01-16
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Hongxia Xu; Alex Aravanis; Shengrong Lin
- Web page: https://patentgrep.com/patent/US10865444B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure relates to the field of molecular biology and more specifically to methods for capturing, amplifying and sequencing target polynucleotides on a solid surface.

## Claims

### Claim 1

A method for determining a true nucleic acid sequence variant, comprising:
(a) contacting a nucleic acid sample comprising a plurality of target polynucleotides with gene specific forward and reverse primers under conditions sufficient for hybridization, each species of said gene specific forward primer comprising a unique sequence index and an adapter;
(b) amplifying by polymerase chain reaction (PCR) said plurality of target polynucleotides to produce a plurality of amplicons;
(c) directly contacting a plurality of target specific capture primers immobilized on a solid support with said plurality of amplicons under conditions sufficient for hybridization to produce a first plurality of immobilized amplicons, said solid support further comprising a plurality of universal capture primers;
(d) extending said plurality of target specific capture primers to produce a plurality of immobilized extension products complementary to said target polynucleotides, wherein said immobilized extension products comprise a terminal adapter portion capable of hybridizing to said universal capture primers;
(e) annealing said plurality of universal capture primers to said plurality of said immobilized extension products;
(f) amplifying by PCR said plurality of immobilized extension products to produce a second plurality of immobilized amplicons, wherein said second plurality of immobilized amplicons comprises a uniformity of 85% or more;
(g) sequencing said second plurality of immobilized amplicons, and
(h) eliminating random sequence errors for one or more target polynucleotide by comparing three or more nucleotide sequences at a variant position for a target polynucleotide species, wherein said target polynucleotide species are identified by said unique sequence index to thereby determine a true nucleotide sequence variant in said one or more target polynucleotides.

### Claim 2 (depends on 1)

The method of claim 1, wherein said plurality of target polynucleotides comprises 10 ng or less input nucleic acid.

### Claim 3 (depends on 1)

The method of claim 1, wherein said conditions sufficient for hybridization comprise incubation for 10 minutes or less.

### Claim 4 (depends on 1)

The method of claim 1, wherein a mismatch rate of 0.3% or less for a variant nucleotide position is detected.

### Claim 5 (depends on 1)

The method of claim 1, wherein contacting step (a) comprises a first round of PCR amplification with a first gene specific forward and reverse primers and a second round of PCR amplification with a second forward primer complementary to a portion of said first gene specific forward primer, second forward primer comprising said unique sequence index and said adapter.

### Claim 6 (depends on 1)

The method of claim 1, wherein said adapter is complementary to said plurality of universal capture primers.

### Claim 7 (depends on 1)

The method of claim 1, wherein said amplification of said plurality of target polynucleotides comprises asymmetrical PCR.

### Claim 8 (depends on 1)

The method of claim 1, wherein said amplification of said plurality of target polynucleotides comprises multiplex amplification.

### Claim 9 (depends on 8)

The method of claim 8, wherein said multiplex amplification comprises a multiplexicity of 180 or more.

### Claim 10 (depends on 1)

The method of claim 1, wherein said solid support further comprises a second plurality of universal capture primers.

### Claim 11 (depends on 10)

The method of claim 10, wherein said plurality of target specific capture primers further comprise a universal capture primer region.

### Claim 12 (depends on 11)

The method of claim 11, wherein said universal capture primer region has a nucleotide sequence corresponding to said second plurality of universal capture primers.

### Claim 13 (depends on 1)

The method of claim 1, wherein said plurality of target specific capture primers comprise 200 or more different nucleotide sequences.

### Claim 14 (depends on 1)

The method of claim 1, wherein said plurality of target polynucleotides comprise 200 or more different nucleotide sequences.

### Claim 15 (depends on 1)

The method of claim 1, wherein said plurality of target polynucleotides comprise a plurality of genomic nucleic acids.

### Claim 16 (depends on 1)

The method of claim 1, wherein said amplification of said immobilized extension products comprises bridge amplification.

### Claim 17 (depends on 1)

The method of claim 1, wherein said solid support comprises a flow cell.

### Claim 18 (depends on 1)

The method of claim 1, wherein said sequencing comprises 50 cycles.

### Claim 19 (depends on 18)

The method of claim 18, wherein a start-to-finish time comprises 3 hours or less.