# US 11,028,435 B2 · Optimal index sequences for multiplex massively parallel sequencing

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- Number: US 11,028,435 B2
- Document ID: US11028435B2
- Status: Granted
- Kind: B2
- Application number: 15/968,613
- Filed: 2018-05-01
- Published: 2018-11-22
- Granted: 2021-06-08
- Earliest claimed: 2017-05-01
- Assignees: Illumina, Inc.
- Applicants: Ilumina, Inc.
- Inventors: Ryan Kelley; Gordon Bean; Eric Vermaas
- Web page: https://patentgrep.com/patent/US11028435B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6869: Methods for sequencing
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- 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)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- 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 set of oligonucleotides comprising a plurality of subsets of oligonucleotides, wherein:
the set of oligonucleotides is configured to identify sources of nucleic acid samples in multiplex massively parallel sequencing, each of the nucleic acid samples comprising a plurality of nucleic acid molecules;
the set of oligonucleotides comprises a set of index sequences comprising at least 6 different index sequences, each subset of the plurality of subsets of oligonucleotides comprising a plurality of index sequences of the set of index sequences;
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; and
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 2 (depends on 1)

The set of oligonucleotides of claim 1, wherein each subset of the plurality of subsets of oligonucleotides comprises one or more pairs of color-balanced index sequences.

### Claim 3 (depends on 1)

The set of oligonucleotides of claim 1, wherein the set of oligonucleotides comprises (a) double-stranded or Y-shaped sequencing adapters, each strand of each double-stranded or Y-shaped sequencing adapter comprises an index sequence of the set of index sequences or a reverse complement thereof; or (b) pairs of single-stranded oligonucleotides, each pair being provided together in a reagent, each oligonucleotide of a pair comprising an index sequence of the set of index sequences or a reverse complement thereof.

### Claim 4 (depends on 3)

The set of oligonucleotides of claim 3, wherein (a) each Y-shaped or double-stranded sequencing adapter comprises a first strand comprising a first index sequence selected from a first subset of the set of index sequences and a second strand comprising a second index sequence selected from a second subset of the set of index sequences (or reverse complements of the second subset); or (b) each pair of oligonucleotides comprises a first oligonucleotide comprising a first index sequence selected from a first subset of the set of index sequences and a second oligonucleotide comprising a second index sequence selected from a second subset of the set of index sequences (or reverse complements of the second subset).

### Claim 5 (depends on 4)

The set of oligonucleotides of claim 4, wherein the first strand of each Y-shaped sequencing adapter comprises a P5 flow cell amplification primer binding site, and the second strand of each Y-shaped sequencing adapter comprises a P7′ flow cell amplification primer binding site.

### Claim 6 (depends on 4)

The set of oligonucleotides of claim 4, 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 thereof; the nth 10-mer in SEQ ID NO: 12 and nth 10-mer in SEQ ID NO: 13 or a reverse complement thereof; the nth 10-mer in SEQ ID NO: 14 and nth 10-mer in SEQ ID NO: 15 or a reverse complement thereof; the nth 10-mer in SEQ ID NO: 16 and nth 10-mer in SEQ ID NO: 17 or a reverse complement thereof; or the nth 10-mer in SEQ ID NO: 18 and nth 10-mer in SEQ ID NO: 19 or a reverse complement thereof.

### Claim 7 (depends on 1)

The set of oligonucleotides 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 is not less than a second criterion value.

### Claim 8 (depends on 7)

The set of oligonucleotides of claim 7, wherein the second criterion value is larger than the first criterion value.

### Claim 9 (depends on 1)

The set of oligonucleotides of claim 1, wherein an oligonucleotide of the set of oligonucleotides comprises a primer having an index sequence on its 3′ end and an index sequence on its 5′ end.

### Claim 10 (depends on 1)

The set of oligonucleotides of claim 1, wherein the set of index sequences is comprised in Y-shaped sequencing adapters, each Y-shaped sequencing adapter comprising an index sequence on only one strand.

### Claim 11 (depends on 1)

The set of oligonucleotides 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 12 (depends on 11)

The set of oligonucleotides of claim 11, wherein the edit distance is a modified Levenshtein distance where end gaps are assigned no penalty.

### Claim 13 (depends on 11)

The set of oligonucleotides of claim 11, wherein:
each index sequence of the set of index sequences has 8 bases;
the first criterion value is 3; and
the third criterion is 2.

### Claim 14 (depends on 11)

The set of oligonucleotides of claim 11, 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 15 (depends on 1)

The set of oligonucleotides of claim 1, wherein the set of index sequences comprising at least 96 different index sequences.

### Claim 16 (depends on 1)

The set of oligonucleotides of claim 1, wherein the set of oligonucleotides are provided in a container comprising multiple separate compartments, and wherein each compartment contains (a) a first plurality of oligonucleotides comprising a first index sequence of the set of index sequences and (b) a second plurality of oligonucleotides comprising a second index sequence of the set of index sequences, an ordered combination of (a) and (b) in the compartment being different from ordered combinations of (a) and (b) in any other compartments.

### Claim 17 (depends on 16)

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

### Claim 18 (depends on 16)

The set of oligonucleotides of claim 16, wherein each index sequence comprised in the first plurality of oligonucleotides is selected from a first subset of the set of index sequences, and each index sequence comprised in the second plurality of oligonucleotides is selected from a second subset of the set of index sequences, the first subset not overlapping the second sub set.

### Claim 19 (depends on 18)

The set of oligonucleotides of claim 18, wherein a Hamming distance between any two index sequences in the first subset or between any two index sequences in the second subset is not less than a second criterion value.

### Claim 20 (depends on 18)

The set of oligonucleotides of claim 18, wherein the second criterion value is larger than the first criterion value.

### Claim 21

A method for making a plurality of oligonucleotides for multiplex massively parallel sequencing, the method comprising:
(a) selecting a set of index sequences from a pool of different index sequences, wherein
the set of index sequences comprise at least 6 different sequences;
a Hamming distance between any two index sequences in the set of index sequences is not less than a first criterion value, wherein the first criterion value is at least 2, and
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; and
(b) synthesizing the plurality of oligonucleotides comprising the set of index sequences.

### Claim 22 (depends on 21)

The method of claim 21, wherein step (a) comprises:
(i) selecting a candidate set of index sequences from the pool of index sequences;
(ii) separating the selected candidate set into a plurality of groups of color-balanced pairs of index sequences; and
(iii) partitioning each group into two subgroups of color-balanced pairs using a bipartite graph matching algorithm, wherein each color-balanced pair is a node in the bipartite graph.

### Claim 23 (depends on 22)

The method of claim 22, wherein two nodes are connected if the Hamming distance between index sequences of the two nodes is less than a second criterion value, wherein the second criterion value is larger than the first criterion value.

### Claim 24 (depends on 22)

The method of claim 22, wherein step (i) comprises:
(1) adding to the candidate set a randomly chosen pair of color-balanced index sequences from the pool of index sequences, wherein the pool includes all possible n-mers;
(2) sorting index sequences remaining in the pool of index sequences based on minimum Hamming distance to members in the candidate set;
(3) removing any remaining index sequence whose minimum Hamming distance to the members in the candidate set is less than the first criterion value or minimum edit distance to the members in the candidate set is less than a third criterion; and
repeating steps (1)-(3) to maximize a size of the candidate set.

### Claim 25 (depends on 22)

The method of claim 22, wherein step (ii) comprises:
(1) randomly selecting a seed for each of the plurality of groups; and
(2) greedily expanding each of the plurality of groups.

### Claim 26

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 selecting a set of index sequences to be incorporated in a set of oligonucleotides configured to be used to in multiplex massively parallel sequencing, said program code comprising:
(a) code for adding to a candidate set of index sequences a randomly chosen pair of color-balanced index sequences from a pool of different 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;
(b) code for sorting index sequences remaining in the pool of index sequences based on minimum Hamming distance to members in the candidate set;
(c) code for removing any remaining index sequence whose minimum Hamming distance to the members in the candidate set is less than a first criterion value or minimum edit distance to the members in the candidate set is less than a second criterion value;
(d) code for repeating (a)-(c) to maximize a size of the candidate set; and
(e) code for selecting from the candidate set the set of index sequences to be incorporated into the set of oligonucleotides configured to be used in multiplex massively parallel sequencing.

### Claim 27 (depends on 26)

The computer program product of claim 26, said program code further comprising:
code for separating the candidate set into a plurality of groups; and
code for partitioning each group into two subgroups using a bipartite graph matching algorithm, wherein each index sequence is a node.