Sequence adapter manufacture and use

Patent metadata

Field	Value
Publication number	US-11352662-B2
Application number	US-201816479473-A
Country	US
Kind code	B2
Filing date	Jan 22, 2018
Priority date	Jan 20, 2017
Publication date	Jun 7, 2022
Grant date	Jun 7, 2022

Abstract

Technology provided herein relates in part to methods, processes, machines and apparatuses for determining sequences of nucleotides for nucleic acid templates in a nucleic acid sample. The technology provide herein also relates in part to methods, processes, machines and apparatuses for counting nucleic acid templates. Nucleic acid templates of a sample are tagged with nonrandom oligonucleotide adapters that include predetermined non-randomly generated sequences. The use of these nonrandom oligonucleotide adapters provides an efficient method to reduce sequencing errors, and increase the sensitivity of detection of low-frequency single nucleotide alterations.

First claim

What is claimed is:

1. A method for determining the sequence of double-stranded nucleic acid templates in a nucleic acid sample, wherein the nucleic acid templates are of different double-stranded nucleic acid template species, the method comprising:
(a) contacting the double-stranded nucleic acid templates with a predetermined discrete set of partially double-stranded nonrandom oligonucleotide adapter species under ligation conditions, thereby generating nonrandom oligonucleotide adapter-ligated nucleic acid templates, wherein:
each of the nonrandom oligonucleotide adapter species comprises a first oligonucleotide species and a second oligonucleotide species;
each of the first oligonucleotide species comprises a polynucleotide A species and a polynucleotide B species and each of the second oligonucleotide species comprises a polynucleotide B′ species and a polynucleotide A′ species;
each of the polynucleotide B species and the polynucleotide B′ species are predetermined, are non-randomly generated and are about 4 to about 20 consecutive nucleotides in length;
there are 999 or fewer polynucleotide B species and each polynucleotide B′ species is a reverse complement of a polynucleotide B species;
each polynucleotide A species is not a reverse complement of polynucleotide A′ species;
the ratio of the double-stranded nucleic acid template species to the polynucleotide B species is greater than 1,000 to 1;
the polynucleotide B species anneal to the complementary polynucleotide B′ species and the polynucleotide A′ species does not anneal to the polynucleotide A species;
(b) amplifying the adapter-ligated nucleic acid templates, thereby generating amplicons; and sequencing all or a portion of each amplicon using a sequencer, thereby generating thousands to millions of sequence reads;
(c) obtaining a list of polynucleotide B species and polynucleotide B′ species of the nonrandom oligonucleotide adapter species provided for ligation with the nucleic acid template species;
(d) from the thousands to millions of sequence reads, determining the sequence of the polynucleotide B species or polynucleotide B′ species of the nonrandom oligonucleotide adapter-ligated nucleic acid templates;
(e) comparing the sequence of the polynucleotide B species or polynucleotide B′ species of step (d) to the sequences of the polynucleotide B and the polynucleotide B′ species on the list obtained from step (c); and
(f) removing from the determination of the count of nucleic acid template species, nonrandom oligonucleotide adapter-ligated nucleic acid templates that comprise B species or B′ species sequences that are not identical to a polynucleotide B species or polynucleotide B′ species sequence on the obtained list, or
assigning a weight to the nonrandom oligonucleotide adapter-ligated nucleic acid template based on the identity of the polynucleotide B species or polynucleotide B′ species in the adapter-ligated nucleic acid templates as compared to the B species or B′ species sequence on the obtained list, where the assigned weight is considered in determining at least one base call;

thereby determining the sequence of the double-stranded nucleic acid templates in the nucleic acid sample.

2. The method of claim 1 , wherein the partially double stranded double-stranded nonrandom oligonucleotide adapter species is a Y adapter or a hairpin adapter.

3. The method of claim 1 , wherein the polynucleotide B species and the polynucleotide B′ species are non-degenerate.

4. The method of claim 1 , wherein
each of the first oligonucleotide species comprises a polynucleotide C species between polynucleotide A and the polynucleotide B species;
each of the second oligonucleotide species comprises a polynucleotide C′ species between polynucleotide A′ and the polynucleotide B′ species;
each polynucleotide C′ species is the reverse complement of the polynucleotide C species; and
the polynucleotide C species anneal to complementary polynucleotide C′ species.

5. The method of claim 4 , wherein each of the polynucleotide C species comprises the same nucleotide sequence or wherein the polynucleotide C species comprises at least two different nucleotide sequences.

6. The method of claim 1 , wherein amplifying the nonrandom oligonucleotide adapter-ligated nucleic acid templates generates double-stranded amplicons, and sequencing comprises sequencing all or a portion of each strand of the amplicons.

7. The method of claim 1 , wherein the nonrandom oligonucleotide adapter-ligated nucleic acid templates are amplified by a process comprising at least one of linear amplification, exponential amplification, or isothermal amplification.

8. The method of claim 1 , wherein each adapter-ligated nucleic acid template comprises one nonrandom oligonucleotide adapter at a first end and a standard sequencing adapter at a second end, or
wherein the each of the adapter-ligated nucleic acid templates comprises a first nonrandom oligonucleotide adapter at a first end and a second nonrandom oligonucleotide adapter at a second end.

9. The method of claim 1 , wherein the nucleic acid templates comprise at least one blunt end, or
wherein the nucleic acid templates are sheared double-stranded DNA templates.

10. The method of claim 1 , comprising blunt-ending the nucleic acid templates before contacting the nucleic acid templates with the nonrandom oligonucleotide adapter species.

11. The method of claim 1 , wherein the nonrandom oligonucleotide adapter species comprise a blunt end.

12. The method of claim 1 , wherein the double-stranded nucleic acid templates or the partially double-stranded nonrandom oligonucleotide adapter species comprise a ligation linker.

13. The method of claim 1 ,
wherein the method further comprises detecting presence of a single nucleotide alteration in the nucleic acid template from the thousands to millions of sequence reads, wherein the single nucleotide alteration is present at a frequency of 5 percent or lower.

14. The method of claim 1 , comprising providing a base call, wherein each base call represents a single nucleotide located at a single nucleotide position in the nucleic acid template.

15. The method of claim 1 , wherein generating nonrandom oligonucleotide adapter-ligated nucleic acid templates is performed by contacting 20-40 ng double-stranded nucleic acid templates of the nucleic acid sample with 50-500 nM partially double-stranded nonrandom oligonucleotide adapter species under ligation conditions.

16. A method for counting the number of nucleic acid template species for a nucleic acid sample, comprising:
(a) contacting double-stranded nucleic acid templates of the nucleic acid sample with a predetermined discrete set of partially double-stranded nonrandom oligonucleotide adapter species under ligation conditions, wherein the double-stranded nucleic acid templates are of different double-stranded nucleic acid template species,
thereby generating nonrandom oligonucleotide adapter-ligated nucleic acid templates, wherein:
each of the nonrandom oligonucleotide adapter species comprises a first oligonucleotide species and a second oligonucleotide species;
each of the first oligonucleotide species comprises a polynucleotide A species and a polynucleotide B species and each of the second oligonucleotide species comprises a polynucleotide B′ species and a polynucleotide A′ species;
each of the polynucleotide B species and the polynucleotide B′ species are predetermined, are non-randomly generated and are about 4 to about 20 consecutive nucleotides in length;
there are 999 or fewer polynucleotide B species a

Assignees

• Sequenom Inc

Inventors

• Jensen Taylor
• Ellison Christopher

Classifications

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12N15/1068

  Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis · CPC title

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title