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US 2019/0292575 A1Published application
Molecular tagging methods and sequencing libraries
AssigneeThe Translational Genomics Research Institute
InventorsMuhammed Murtaza, Maria de las Nieves Perdigones Borderias
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Abstract
The invention provides a tagged sequencing library and methods for tagging low abundance target sequences and generating a sequencing library for detecting low abundance target sequences.
Claims25
A method of adding an oligonucleotide tag to a nucleic acid sequence, the method comprising the steps of:
- annealing a first universal primer to the nucleic acid sequence, wherein the first universal primer is complementary to a sequence of interest on the nucleic acid sequence;
- linearly amplifying the nucleic acid sequence;
- and
- ligating an adapter oligonucleotide to the 3′-end of the nucleic acid sequence, wherein the adaptor oligonucleotide comprises:
- a stem-loop intramolecular nucleotide base pairing;
- a hydroxyl group at the 3′-end;
- a phosphate at the 5′-end;
- a first random region complementary to the nucleic acid sequence;
- and
- a second random region in the loop comprising a molecular barcode.
7 more elements
A method of producing a sequencing library, the method comprising the steps of:
- ligating an adapter oligonucleotide to the 3′-end of a nucleic acid sequence to produce a hybrid sequence comprising a sequence of interest on the nucleic acid sequence and the adapter oligonucleotide, wherein the adaptor oligonucleotide comprises:
- a stem-loop intramolecular nucleotide base pairing forming a loop;
- a hydroxyl group at the 3′-end;
- a phosphate at the 5′-end;
- a first random region complementary to the nucleic acid sequence;
- and
- a second random region in the loop comprising a molecular barcode;
- and
- amplifying the hybrid sequence with a first set of primers.
6 more elements
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Dependencies are inferred from the claim text.