Methods of Improving Unique Molecular Index Ligation Efficiency
AssigneeIllumina, Inc.
InventorsLouise Fraser, Natalie Morrell, Ana Rita Borba, Niall Anthony Gormley, Ming-Hsiang Lee, Sorena Rahmanian, Jennifer Lococo, Danny Chou
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Abstract
The disclosed embodiments concern methods for determining sequences of interest using improved unique molecular indexes (UMIs) that are uniquely associable with individual polynucleotide fragments. Pools of adapters comprising UMIs are also provided.
Claims37
A method for sequencing source nucleic acid molecules from a sample using a pool of adapters, wherein the adapters each comprise a unique molecular identifier (UMI), wherein each UMI comprises an oligonucleotide sequence comprising the sequence 5′-XNYZ-3′, wherein the 3′ end of the UMI ends with YZ, wherein YZ does not correspond to GA, GC, or GG, wherein N is an integer greater than or equal to 1 and each X is the same or different, wherein each adapter is attached to a solid support, and wherein each UMI is an oligonucleotide sequence that can be used to identify an individual molecule of a source nucleic acid fragment in the sample, comprising:
- a) fragmenting source nucleic acid molecules into nucleic acid fragments;
- b) applying the pool of adapters to source nucleic acid fragments in the sample;
- c) amplifying both strands of the source nucleic acid-adapter products to obtain a plurality of amplified polynucleotides;
- d) sequencing the plurality of amplified polynucleotides, thereby obtaining a plurality of reads each associated with a UMI;
- e) identifying a plurality of UMIs associated with the plurality of reads;
- f) determining sequences of the source nucleic acid fragments in the sample using the plurality of reads obtained in (c) and the plurality of UMIs identified in (d).
A method for sequencing source nucleic acid molecules from a sample using a pool of adapters, wherein the adapters each comprise a primer, a primer binding site, an index sequence, and/or a barcode, wherein each adapter comprises an oligonucleotide sequence comprising the sequence 5′-XNYZ-3′, wherein the 3′ end of the adapter ends with YZ, wherein YZ does not correspond to GA, GC, or GG, wherein N is an integer greater than or equal to 1 and each X is the same or different, wherein each adapter comprises an oligonucleotide sequence comprising the sequence 5′-XNYZ-3′, wherein the 3′ end of the adapter ends with YZ, wherein YZ does not correspond to GA, GC, or GG, and wherein N is an integer greater than or equal to 1 and each X is the same or different, and wherein each adapter is attached to a solid support, comprising:
- a) fragmenting source nucleic acid molecules into nucleic acid fragments;
- b) applying the pool of adapters to source nucleic acid fragments in the sample;
- c) amplifying both strands of the source nucleic acid-adapter products to obtain a plurality of amplified polynucleotides;
- and
- d) sequencing the plurality of amplified polynucleotides.
A pool of adapters, wherein the adapters each comprise a unique molecular identifier (UMI), wherein each UMI comprises an oligonucleotide sequence comprising the sequence 5′-XNYZ-3′, wherein the 3′ end of the UMI ends with YZ, wherein YZ does not correspond to GA, GC, or GG, wherein N is an integer greater than or equal to 1 and each X is the same or different, and wherein each adapter is attached to a solid support.
A pool of adapters, wherein the adapters each comprise a primer, a primer binding site, an index sequence, and/or a barcode, wherein each adapter comprises an oligonucleotide sequence comprising the sequence 5′-XNYZ-3′, wherein the 3′ end of the adapter ends with YZ, wherein YZ does not correspond to GA, GC, or GG, wherein N is an integer greater than or equal to 1 and each X is the same or different, and wherein each adapter is attached to a solid support.
Dependencies are inferred from the claim text.