# US 11,028,436 B2 · Universal short adapters for indexing of polynucleotide samples

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- Number: US 11,028,436 B2
- Document ID: US11028436B2
- Status: Granted
- Kind: B2
- Application number: 15/973,210
- Filed: 2018-05-07
- Published: 2018-11-22
- Granted: 2021-06-08
- Earliest claimed: 2017-05-08
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Tatjana Singer; Ryan Kelley; Gordon Bean; Eric Vermaas
- Web page: https://patentgrep.com/patent/US11028436B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6869: Methods for sequencing
- G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- G16B 25/20: Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
- 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
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 6)

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.

### Claim 8 (depends on 7)

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.

### Claim 9 (depends on 7)

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.

### Claim 10 (depends on 8)

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.

### Claim 11 (depends on 10)

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.

### Claim 12 (depends on 11)

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

### Claim 13 (depends on 1)

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.

### Claim 14 (depends on 13)

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.

### Claim 15 (depends on 1)

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.

### Claim 16 (depends on 1)

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.

### Claim 17 (depends on 5)

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.

### Claim 18 (depends on 5)

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

### Claim 19 (depends on 1)

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

### Claim 20 (depends on 13)

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.

### Claim 21 (depends on 20)

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

### Claim 22 (depends on 1)

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

### Claim 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 a transposase and two transposon end compositions;
(c) incubating the double-stranded nucleic acid molecules with the transposome complexes to obtain double-stranded nucleic acid fragments, wherein the double-stranded nucleic acid fragments comprise, at one or both ends, sequences transposed from the transposon end compositions;
(d) contacting a plurality of index primers with the double-stranded nucleic acid fragments to generate a plurality of index-fragment polynucleotides, wherein
index primers contacted with double-stranded nucleic acid fragments derived from each sample comprise an index sequence or a combination of index sequences that is uniquely associated with that sample, and
the index sequence or the combination of index sequences is selected from a set of index sequences;
(e) pooling the plurality of index-fragment polynucleotides;
(f) sequencing the pooled index-fragment polynucleotides, thereby obtaining 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
(g) using the index reads to determine the target reads' sources of samples.

### Claim 24

A computer program product comprising a non-transitory machine readable medium storing program code that, when executed by one or more processors of a computer system, causes the computer system to implement a method for sequencing target nucleic acids derived from a plurality of samples, said program code comprising:
(a) code for receiving a plurality of index reads and a plurality of target reads of target sequences obtained from target nucleic acids derived from the plurality of samples, wherein
each target read comprises a target sequence obtained from a target nucleic acid derived from a sample of the plurality of samples,
each index read comprises an index sequence obtained from a target nucleic acid derived from a sample of the plurality of samples, the index sequence being selected from a set of index sequences,
each target read is associated with at least one index read,
each sample of the plurality of samples is uniquely associated with one or more index sequences of the 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) code for identifying, among the plurality of target reads, a subset of target reads associated with index reads aligned to at least one index sequence uniquely associated with a particular sample of the plurality of samples; and
(c) code for determining a target sequence for the particular sample based on the identified subset of target reads.

### Claim 25 (depends on 24)

The computer program product of claim 24, 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 base or a cytosine base, and (ii) a guanine base, a thymine base, or a uracil base.