Universal short adapters with variable length non-random unique molecular identifiers

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.

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.

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.

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.

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.