Universal short adapters for indexing of polynucleotide samples

Patent metadata

Field	Value
Publication number	US-11028436-B2
Application number	US-201815973210-A
Country	US
Kind code	B2
Filing date	May 7, 2018
Priority date	May 8, 2017
Publication date	Jun 8, 2021
Grant date	Jun 8, 2021

Abstract

The disclosed embodiments concern index oligonucleotides configured to identify sources of samples of nucleic acids and methods, apparatus, systems and computer program products for identifying and making the index oligonucleotides. In some implementations, the index oligonucleotides include a set of index sequences, a Hamming distance between any two index sequences of the set of index sequences meeting one or more criteria. System, apparatus, and computer program products are also provided for determining a sequence of interest using the index oligonucleotides.

First claim

What is claimed is:

1. A method for sequencing target nucleic acids derived from a plurality of samples, comprising
(a) contacting a plurality of index polynucleotides with target nucleic acids derived from the plurality of samples to generate a plurality of index-target polynucleotides, wherein
index polynucleotides contacted with target nucleic acids derived from each sample comprise an index sequence or a combination of index sequences that is uniquely associated with that sample,
the index sequence or the combination of index sequences is selected from a set of index sequences, and
a Hamming distance between any two index sequences of the set of index sequences is not less than a first criterion value, wherein the first criterion value is at least 2;

(b) pooling the plurality of index-target polynucleotides;
(c) sequencing the pooled index-target polynucleotides to obtain a plurality of index reads of index sequences and a plurality of target reads of target sequences, each target read being associated with at least one index read; and
(d) using the index reads to determine the target reads' sources of samples.

2. The method of claim 1 , wherein the set of index sequences comprises a plurality of pairs of color-balanced index sequences, wherein any two bases at corresponding sequence positions of each pair of color-balanced index sequences include both (i) an adenine (A) base or a cytosine (C) base, and (ii) a guanine (G) base, a thymine (T) base, or a uracil (U) base.

3. The method of claim 1 , wherein the set of index sequences comprises at least 6 different index sequences.

4. The method of claim 1 , wherein using the index reads to determine the target reads' sources of samples comprises:
obtaining, for each index read, alignment scores with respect to the set of index sequences, each alignment score indicating similarity between a sequence of the index read and an index sequence of the set of index sequences;
determining that a particular index read is aligned to a particular index sequence based on the alignment scores; and
determining that a target read associated with the particular index read is derived from a sample uniquely associated with the particular index sequence.

5. The method of claim 1 , wherein an edit distance between any two index sequences of the set of index sequences is not less than a third criterion value.

6. The method of claim 1 , wherein the plurality of index polynucleotides comprises a plurality of index primers comprising index sequences of the set of index sequences.

7. The method of claim 6 , wherein the target nucleic acids derived from the plurality of samples comprise nucleic acids with universal adapters covalently attached to one or both ends.

8. The method of claim 7 , wherein the contacting the plurality of index polynucleotides with the target nucleic acids derived from the plurality of samples comprises:
hybridizing the plurality of index primers to the universal adapters covalently attached to one or both ends of the nucleic acids; and
extending the plurality of index primers to obtain a plurality of index-adapter-target polynucleotides.

9. The method of claim 7 , wherein the universal adapters are selected from the group consisting of double-stranded adapters, Y-shaped adapters, single-stranded adapters, and hairpin adapters.

10. The method of claim 8 , wherein the universal adapters and the target nucleic acids are double stranded, and hybridizing the plurality of index primers to the universal adapters comprises hybridizing the plurality of index primers to both strands of the universal adapters.

11. The method of claim 10 , wherein a first index primer hybridized to a first strand of a particular universal adapter comprises a first index sequence selected from a first subset of the set of index sequences, and a second index primer hybridized to a second strand of the particular universal adapter comprise a second index sequence selected from a second subset of the set of index sequences.

12. The method of claim 11 , wherein the first and the second index sequences respectively are:
the n th 10-mer in SEQ ID NO: 10 and n th 10-mer in SEQ ID NO: 11 or a reverse complement of SEQ ID NO: 11;
the n th 10-mer in SEQ ID NO: 12 and n th 10-mer in SEQ ID NO: 13 or a reverse complement of SEQ ID NO: 13;
the n th 10-mer in SEQ ID NO: 14 and n th 10-mer in SEQ ID NO: 15 or a reverse complement of SEQ ID NO: 15;
the n th 10-mer in SEQ ID NO: 16 and n th 10-mer in SEQ ID NO: 17 or a reverse complement of SEQ ID NO: 17; or
the n th 10-mer in SEQ ID NO: 18 and n th 10-mer in SEQ ID NO: 19 or a reverse complement of SEQ ID NO: 19.

13. The method of claim 1 , wherein the plurality of index polynucleotides comprises sample-specific double-stranded adapters, each strand of said adapters comprising an index sequence of the set of index sequences.

14. The method of claim 13 , wherein the sample-specific adapters are selected from the group consisting of: Y-shaped adapters having a complementary double-stranded region and a mismatched single-stranded region, single-stranded adapters, and hairpin adapters.

15. The method of claim 1 , wherein the contacting the plurality of index polynucleotides with the target nucleic acids comprises attaching at least one of the plurality of index polynucleotides to one end or both ends of the target nucleic acids.

16. The method of claim 1 , wherein the set of index sequences comprises a plurality of non-overlapping subsets of index sequences, a Hamming distance between any two index sequences in any subset being not less than a second criterion value, wherein the second criterion value is larger than the first criterion value.

17. The method of claim 5 , wherein:
each index sequence of the set of index sequences has 10 bases;
the first criterion value is 4; and
the third criterion is 3.

18. The method of claim 5 , wherein the edit distance is a modified Levenshtein distance where end gaps are assigned no penalty.

19. The method of claim 1 , wherein the set of index sequences comprises at least 96 different index sequences.

20. The method of claim 13 , wherein a first strand of each sample-specific adapter comprises a first index sequence selected from a first subset of the set of index sequences, and a second strand of the sample-specific adapter comprises a second index sequence selected from a second subset of the set of index sequences.

21. The method of claim 20 , wherein the first and the second index sequences respectively are:
the n th 10-mer in SEQ ID NO: 10 and n th 10-mer in SEQ ID NO: 11 or a reverse complement of SEQ ID NO: 11;
the n th 10-mer in SEQ ID NO: 12 and n th 10-mer in SEQ ID NO: 13 or a reverse complement of SEQ ID NO: 13;
the n th 10-mer in SEQ ID NO: 14 and n th 10-mer in SEQ ID NO: 15 or a reverse complement of SEQ ID NO: 15;
the n th 10-mer in SEQ ID NO: 16 and n th 10-mer in SEQ ID NO: 17 or a reverse complement of SEQ ID NO: 17; or
the n th 10-mer in SEQ ID NO: 18 and n th 10-mer in SEQ ID NO: 19 or a reverse complement of SEQ ID NO: 19.

22. The method of claim 1 , wherein each index sequence has 10 or fewer bases.

23. A method for sequencing target nucleic acids derived from a plurality of samples, comprising:
(a) providing a plurality of double-stranded nucleic acid molecules derived from the plurality of samples;
(b) providing a plurality of transposome complexes, wherein each transposome complex comprises

Assignees

• Illumina Inc

Inventors

• Singer Tatjana
• Kelley Ryan
• Bean Gordon
• Vermaas Eric

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• G16B30/10 Primary

  Sequence alignment; Homology search · CPC title