# US 11,898,198 B2 · Universal short adapters with variable length non-random unique molecular identifiers

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- Number: US 11,898,198 B2
- Document ID: US11898198B2
- Status: Granted
- Kind: B2
- Application number: 17/890,208
- Filed: 2022-08-17
- Published: 2023-08-03
- Granted: 2024-02-13
- Earliest claimed: 2017-09-15
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Chen Zhao; Kevin Wu; Han-Yu Chuang; Jennifer Lococo; Alex So; Dwight Baker; Tatjana Singer
- Web page: https://patentgrep.com/patent/US11898198B2

## CPC

- C12Q 1/6855: Ligating adaptors
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B 20/20: Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
- G16B 25/20: Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B 30/10: Sequence alignment; Homology search
- G16B 35/10: Design of libraries
- G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- C12Q 2525/191: incorporating an adaptor
- C12Q 2525/197: incorporating a spacer/coupling moiety
- C12Q 2535/122: Massive parallel sequencing
- C12Q 2600/16: Primer sets for multiplex assays
- C12Q 2600/166: Oligonucleotides used as internal standards, controls or normalisation probes
- 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 methods, systems and computer program products for determining sequences of interest using unique molecular indexes (UMIs) that are uniquely associable with individual polynucleotide fragments, including sequences with low allele frequencies or long sequence length. In some implementations, the UMIs include variable-length nonrandom UMIs (vNRUMIs). Methods and systems for making and using sequencing adapters comprising vNRUMIs are also provided.

## Claims

### Claim 1

A set of sequencing adapters comprising a plurality of double-stranded polynucleotides, wherein:
each double-stranded polynucleotide comprises a double-stranded hybridized region, a single-stranded 5′ arm, a single-stranded 3′ arm, and at least one variable-length, nonrandom unique molecular index (vNRUMI);
variable-length, nonrandom unique molecular indices (vNRUMIs) of the set of sequencing adapters form a set of vNRUMIs configured to identify individual nucleic acid molecules in a sample for multiplex massively parallel sequencing;
the set of vNRUMIs comprises sequences having two or more molecular lengths;
an edit distance between any two vNRUMIs of the set of vNRUMIs is not less than a first criterion value, wherein the first criterion value is at least two; and
edit distances among the vNRUMIs in the set of vNRUMIs are maximized by a greedy approach.

### Claim 2 (depends on 1)

The set of sequencing adapters of claim 1, wherein the set of vNRUMIs comprises vNRUMIs of 6 nucleotides and vNRUMIs of 7 nucleotides.

### Claim 3 (depends on 1)

The set of sequencing adapters of claim 1, wherein the first criterion value is at least three.

### Claim 4 (depends on 1)

The set of sequencing adapters of claim 1, wherein the double-stranded hybridized region comprises a sequence of SEQ ID NO: 1 (AGATGTGTATAAGAGACAG).

### Claim 5 (depends on 1)

The set of sequencing adapters of claim 1, wherein the double-stranded hybridized region comprises a sequence of SEQ ID NO: 2 (CTGTCTCTTATACACATCT).

### Claim 6 (depends on 1)

The set of sequencing adapters of claim 1, wherein the single-stranded 5′ arm comprises a first primer binding sequence.

### Claim 7 (depends on 6)

The set of sequencing adapters of claim 6, wherein the first primer binding sequence is a sequence of SEQ ID NO: 3 (TCGTCGGCAGCGTC).

### Claim 8 (depends on 1)

The set of sequencing adapters of claim 1, wherein the single-stranded 3′ arm comprises a second primer binding sequence.

### Claim 9 (depends on 8)

The set of sequencing adapters of claim 8, wherein the second primer binding sequence is a sequence of SEQ ID NO: 5 (CCGAGCCCACGAGAC).

### Claim 10 (depends on 1)

The set of sequencing adapters of claim 1, wherein the double-stranded polynucleotide comprises a vNRUMI on one strand of the double-stranded hybridized region and a reverse complement of the vNRUMI on another strand of the double-stranded hybridized region.

### Claim 11 (depends on 1)

The set of sequencing adapters of claim 1, wherein the double-stranded polynucleotide comprises a vNRUMI on the single-stranded 5′ arm.

### Claim 12 (depends on 1)

The set of sequencing adapters of claim 1, wherein the double-stranded polynucleotide comprises a vNRUMI on the single-stranded 3′ arm.

### Claim 13 (depends on 1)

The set of sequencing adapters of claim 1, wherein the set of vNRUMIs comprises: CACATGA, GGTTAC, TTGCCAG, AACCGC, ATGGTG, CTAGAAC, AGAATAG, TCAACTC, GTTCGGA, AAGACA, ACATTC, ACCAAG, CAGTAG, CCACCA, CTTGGC, GCCTGA, TGAGGA, TGTCCG, TAGCGTA, AGTCGAC, GTACACG, CCTATTG, TCGGAGA, GCTGTCA, TCCTTGC, GTGAGTC, TAATGCG, AGGCTCA, AACTAAC, GATGAAG, ATAACCA, TATGTTC, GGATTGA, GGCCATA, AACGTA, AATGAG, ACAGCG, ACGCAC, ACTAGA, AGAAGC, AGACTG, AGTGCA, ATTACG, CAACAC, CAGGTC, CATTGA, CCGATA, CCTAAC, CCTGTG, CGAACG, CGCAGA, CGCTTC, CTCCAG, GAAGTG, GACAAC, GAGCTA, GCACAG, GCGTTG, GGCATG, GTAACA, GTATGC, GTCCTC, GTGGAC, GTTGTA, TACCTG, TACTCA, TCAATG, TCACGC, TCGGCA, TGATAG, TGCCAC, TGTGTC, TCAGAAG, TTGTGAC, GATAGGC, TGAGCTG, ACGTTAC, TTGAACA, TATGGCA, TGTATAC, CACCTAC, ACGAGCA, GCGAATG, GCATACA, TCCTACG, TGTCATG, AGTGGTA, CGGTAAG, CCATAGC, CTTCCTG, GTTAGCG, CTCGATG, TTCGAGC, AAGTCCA, CTAAGGA, ATAAGTG, CTTGAGA, CCTCATA, TGCACCA, AGAGACG, GAACCTC, ATTGTCG, GAACGAG, ATAGCAG, CTAGTTA, TCGTGTG, AGGATTC, GTGCAAC, TACATAG, CTACTGC, GCAGTTC, TAGACGC, TTACCGA, CGGTGTA, CAATTAG, ACCGTTG, AAGGATG, GAGTCAG, ATGTAGC, and ATTCACA.

### Claim 14

A set of sequencing adapters comprising a plurality of double-stranded polynucleotides, wherein:
each double-stranded polynucleotide comprises at least one variable-length, nonrandom unique molecular index (vNRUMI);
variable-length, nonrandom unique molecular indices (vNRUMIs) of the set of sequencing adapters form a set of vNRUMIs configured to identify individual nucleic acid molecules in a sample for multiplex massively parallel sequencing; and
the set of vNRUMIs comprises sequences having two or more molecular lengths;
an edit distance between any two vNRUMIs of the set of vNRUMIs is not less than a first criterion value, wherein the first criterion value is at least two; and
edit distances among the vNRUMIs in the set of vNRUMIs are maximized by a greedy approach.

### Claim 15 (depends on 14)

The set of sequencing adapters of claim 14, wherein each double-stranded polynucleotide further comprises a double-stranded hybridized region, a single-stranded 5′ arm, and a single-stranded 3′ arm.

### Claim 16 (depends on 14)

The set of sequencing adapters of claim 14, wherein each double-stranded polynucleotide consists essentially of a double stranded region.

### Claim 17

A method for sequencing nucleic acid molecules from a sample, comprising
(a) applying sequencing adapters to DNA fragments in the sample to obtain DNA-adapter products, wherein
each of the sequencing adapters comprises a double-stranded hybridized region, a single-stranded 5′ arm, a single-stranded 3′ arm, and at least one variable-length, nonrandom unique molecular index (vNRUMI) selected from a set of variable-length, nonrandom unique molecular indices (vNRUMIs) having two or more different molecular lengths,
the set of vNRUMIs is configured to identify individual nucleic acid molecules in a sample for multiplex massively parallel sequencing,
an edit distance between any two vNRUMIs of the set of vNRUMIs is not less than a first criterion value, wherein the first criterion value is at least two, and
edit distances among the vNRUMIs in the set of vNRUMIs are maximized by a greedy approach;
(b) amplifying the DNA-adapter products to obtain a plurality of amplified polynucleotides;
(c) sequencing the plurality of amplified polynucleotides, thereby obtaining a plurality of reads associated with the set of vNRUMIs;
(d) identifying, among the plurality of reads, reads associated with a same vNRUMI; and
(e) determining a sequence of a DNA fragment in the sample using the reads associated with the same vNRUMI.

### Claim 18

A method for sequencing nucleic acid molecules from a sample, comprising
(a) applying sequencing adapters to DNA fragments in the sample to obtain DNA-adapter products, wherein
each of the sequencing adapters comprises at least one variable-length, nonrandom unique molecular index (vNRUMI) selected from a set of variable-length, nonrandom unique molecular indices (vNRUMIs) having two or more different molecular lengths,
the set of vNRUMIs is configured to identify individual nucleic acid molecules in a sample for multiplex massively parallel sequencing, and
an edit distance between any two vNRUMIs of the set of vNRUMIs is not less than a first criterion value, wherein the first criterion value is at least two, and
edit distances among the vNRUMIs in the set of vNRUMIs are maximized by a greedy approach;
(b) amplifying the DNA-adapter products to obtain a plurality of amplified polynucleotides;
(c) sequencing the plurality of amplified polynucleotides, thereby obtaining a plurality of reads associated with the set of vNRUMIs;
(d) identifying, among the plurality of reads, reads associated with a same vNRUMI; and
(e) determining a sequence of a DNA fragment in the sample using the reads associated with the same vNRUMI.

### Claim 19

A set of sequencing adapters comprising a plurality of double-stranded polynucleotides, wherein:
each double-stranded polynucleotide comprises a double-stranded hybridized region, a single-stranded 5′ arm, a single-stranded 3′ arm, and at least one variable-length, nonrandom unique molecular index (vNRUMI);
variable-length, nonrandom unique molecular indices (vNRUMIs) of the set of sequencing adapters form a set of vNRUMIs configured to identify individual nucleic acid molecules in a sample for multiplex massively parallel sequencing;
the set of vNRUMIs comprises sequences having two or more molecular lengths;
an edit distance between any two vNRUMIs of the set of vNRUMIs is not less than a first criterion value, wherein the first criterion value is at least two; and
the set of vNRUMIs comprises: CACATGA, GGTTAC, TTGCCAG, AACCGC, ATGGTG, CTAGAAC, AGAATAG, TCAACTC, GTTCGGA, AAGACA, ACATTC, ACCAAG, CAGTAG, CCACCA, CTTGGC, GCCTGA, TGAGGA, TGTCCG, TAGCGTA, AGTCGAC, GTACACG, CCTATTG, TCGGAGA, GCTGTCA, TCCTTGC, GTGAGTC, TAATGCG, AGGCTCA, AACTAAC, GATGAAG, ATAACCA, TATGTTC, GGATTGA, GGCCATA, AACGTA, AATGAG, ACAGCG, ACGCAC, ACTAGA, AGAAGC, AGACTG, AGTGCA, ATTACG, CAACAC, CAGGTC, CATTGA, CCGATA, CCTAAC, CCTGTG, CGAACG, CGCAGA, CGCTTC, CTCCAG, GAAGTG, GACAAC, GAGCTA, GCACAG, GCGTTG, GGCATG, GTAACA, GTATGC, GTCCTC, GTGGAC, GTTGTA, TACCTG, TACTCA, TCAATG, TCACGC, TCGGCA, TGATAG, TGCCAC, TGTGTC, TCAGAAG, TTGTGAC, GATAGGC, TGAGCTG, ACGTTAC, TTGAACA, TATGGCA, TGTATAC, CACCTAC, ACGAGCA, GCGAATG, GCATACA, TCCTACG, TGTCATG, AGTGGTA, CGGTAAG, CCATAGC, CTTCCTG, GTTAGCG, CTCGATG, TTCGAGC, AAGTCCA, CTAAGGA, ATAAGTG, CTTGAGA, CCTCATA, TGCACCA, AGAGACG, GAACCTC, ATTGTCG, GAACGAG, ATAGCAG, CTAGTTA, TCGTGTG, AGGATTC, GTGCAAC, TACATAG, CTACTGC, GCAGTTC, TAGACGC, TTACCGA, CGGTGTA, CAATTAG, ACCGTTG, AAGGATG, GAGTCAG, ATGTAGC, and ATTCACA.

### Claim 20

A set of sequencing adapters comprising a plurality of double-stranded polynucleotides, wherein:
each double-stranded polynucleotide comprises a double-stranded hybridized region, a single-stranded 5′ arm, a single-stranded 3′ arm, and at least one variable-length, nonrandom unique molecular index (vNRUMI);
the double-stranded hybridized region comprises a sequence of SEQ ID NO: 1 (AGATGTGTATAAGAGACAG) or a sequence of SEQ ID NO: 2 (CTGTCTCTTATACACATCT);
variable-length, nonrandom unique molecular indices (vNRUMIs) of the set of sequencing adapters form a set of vNRUMIs configured to identify individual nucleic acid molecules in a sample for multiplex massively parallel sequencing;
the set of vNRUMIs comprises sequences having two or more molecular lengths; and
an edit distance between any two vNRUMIs of the set of vNRUMIs is not less than a first criterion value, wherein the first criterion value is at least two.