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US 11,891,600 B2Granted
Nucleic acid indexing techniques
AssigneeIllumina, Inc.
InventorsJohn S. Vieceli, Ryan Matthew Kelley
Life of this patent
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Abstract
Presented herein are techniques for indexing of nucleic acid, e.g., for use in conjunction with sequencing. The techniques include generating indexed nucleic acid fragments from an individual sample, whereby the index sequence incorporated into each index site of the nucleic acid fragment is selected from a plurality of distinguishable of index sequences and such that the population of generated nucleic acid fragments represents each index sequence from the plurality. In this manner, the generated indexed nucleic acid fragments from a single sample are indexed with a diverse mix of index sequences that reduce misassignment due to index read errors associated with low sequence diversity.
Claims19
A sample-indexed nucleic acid library preparation, comprising:
- a first nucleic acid library prepared from a first sample, wherein the first nucleic acid library comprises a first plurality of nucleic acid fragments, wherein each nucleic acid fragment of the first plurality comprises at least two different index sequences selected from a first set of index sequences that are all distinguishable from one another, the first set of index sequences comprising a first 5′ subset and a first 3′ subset, wherein the first set of index sequences are arranged on individual nucleic acid fragments of the first plurality such that index sequences of the first 5′ subset are located 5′ of a target sequence and not 3′ of the target sequence, and index sequences of the first 3′ subset are located 3′ of the target sequence and not 5′ of the target sequence, wherein the first plurality comprises more than one combination of the two different index sequences such that one or more individual fragments of the first plurality of nucleic acid fragments have respective different 5′ index sequences and respective different 3′ index sequences relative to one another, wherein more than one fragment of the first plurality has a same combination of the two different index sequences and different target sequences, and wherein the same combination comprises an index sequence of the first 5′ subset and an index sequence of the first 3′ subset;
- and
- a second nucleic acid library prepared from a second sample, wherein the second nucleic acid library comprises a second plurality of nucleic acid fragments, wherein each nucleic acid fragment of the second plurality comprises at least two different index sequences selected from a second set of unique index sequences that are all distinguishable from one another and from the first set of index sequences, the second set of unique index sequences comprising a second 5′ subset and a second 3′ subset, wherein the second set of index sequences are arranged on individual nucleic acid fragments of the second plurality such that index sequences of the second 5′ subset are located 5′ of a target sequence and not 3′ of the target sequence and index sequences of the second 3′ subset are located 3′ of the target sequence and not 5′ of the target sequence wherein the first set of index sequences are associated with the first sample and not the second sample and wherein the second set of index sequences are associated with the second sample and not the first sample, wherein the second plurality comprises more than one combination of the two different index sequences such that one or more individual fragments of the second plurality of nucleic acid fragments have respective different 5′ index sequences and respective different 3′ index sequences relative to one another, and wherein more than one fragment of the second plurality has a same combination of the two different index sequences and different target sequences, wherein the same combination comprises an index sequence of the second 5′ subset and an index sequence of the second 3′ subset.
A sample-indexed nucleic acid library preparation, comprising:
- a first nucleic acid library prepared from a first sample, wherein the first nucleic acid library comprises a first plurality of dual-indexed nucleic acid fragments having respective different target sequences, wherein the first nucleic acid library comprises a first sample 5′ set of different 5′ index sequences and a first sample 3′ set of different 3′ index sequences, and wherein each nucleic acid fragment of the first plurality comprises a 5′ index sequence selected from the first sample 5′ set and a 3′ index selected from the first sample 3′ set, and wherein the different 5′ index sequences and the different 3′ index sequences are randomly combined among the first plurality of dual-indexed nucleic acid fragments such that a plurality of possible combinations of the 5′ index sequence and the 3′ index sequence are represented in the first nucleic acid library and wherein at least one possible combination is present in more than one fragment of the first nucleic acid library;
- and
- a second nucleic acid library prepared from a second sample, wherein the second nucleic acid library comprises a second plurality of dual-indexed nucleic acid fragments having respective different target sequences, wherein the second nucleic acid library comprises a second sample 5′ set of different 5′ index sequences and a second sample 3′ set of different 3′ index sequences, and wherein each nucleic acid fragment of the second plurality comprises a 5′ index sequence selected from the second sample 5′ set and a 3′ index selected from the second sample 3′ set, and wherein the different 5′ index sequences and the different 3′ index sequences are randomly combined among the second plurality of dual-indexed nucleic acid fragments such that a plurality of possible combinations of the 5′ index sequence and the 3′ index sequence are represented in the second nucleic acid library, wherein at least one possible combination is present in more than one fragment of the second nucleic acid library, and wherein the first sample 5′ set of different 5′ index sequences, the first sample 3′ set of different 3′ index sequences, the second sample 5′ set of different 5′ index sequences and the second sample 3′ set of different 3′ index sequences are all distinguishable from one another.
Dependencies are inferred from the claim text.