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US 2019/0194651 A1Published application
Semi-random barcodes for nucleic acid analysis
AssigneeQiagen Sciences, LLC
InventorsYexun Wang, Zhong Wu
Life of this patent
Estimating the term…
Abstract
The present disclosure provides oligonucleotides that comprise semi-random barcode sequences. Such oligonucleotides may be incorporated into reverse transcription primers, PCR primers, or portions of sequencing adapters in preparing sequencing libraries. The resulting sequencing libraries can be used for accurate sequencing, including DNA or RNA counting and mutation detection. Methods and kits for preparing sequencing adapters and sequencing libraries are also provided.
Claims17
A method for preparing a sequencing library, comprising:
- (1) ligating a plurality of partially or fully double-stranded (ds) sequencing adapters to ds DNA fragments of a sample, thereby generating a sequencing library,
- wherein one strand of each of the plurality of the partially or fully ds DNA sequencing adapters comprises, from the 5′ to 3′ direction, a 1st sequence and a 2nd sequence:
- (a) the 1st sequence is a semi-random sequence consisting of (Xmer)n, wherein Xmer is 3-mer, 4-mer, 5-mer, or 6-mer, and n is an integer from 2 to 8, and
- (b) the 2nd sequence is
- (i) at least 10 nucleotides in length,
- (ii) fully or substantially complementary to a target sequence, and
- (iii) the same among the plurality of partially or fully ds sequencing adapters,
- wherein the semi-random sequences are synthesized from a mixture of Xmers with defined sequences,
- wherein different Xmers have 2 or more nucleotide differences from each other, and
- wherein the number of different 1st sequences in the plurality of the partially or fully ds DNA sequencing adapters is at least 100.
7 more elements
A method for preparing a sequencing library, comprising:
- (1) ligating a plurality of partially or fully double-stranded (ds) sequencing adapters to ds DNA fragments of a sample, thereby generating a sequencing library,
- wherein one strand of each of the plurality of the partially or fully ds DNA sequencing adapters comprises, from the 5′ to 3′ direction, a 1st sequence and a 2nd sequence:
- (a) the 1st sequence is a semi-random sequence consisting of (Xmer)n, wherein Xmer is 3-mer, 4-mer, 5-mer, or 6-mer, and n is an integer from 2 to 8, and
- (b) the 2nd sequence is
- (i) at least 10 nucleotides in length,
- (ii) fully or substantially complementary to a target sequence, and
- (iii) the same among the plurality of partially or fully ds sequencing adapters,
- wherein the semi-random sequences are synthesized from a mixture of Xmers with defined sequences,
- wherein the number of different Xmers in the mixture is 2 to 25, and
- wherein the number of different 1st sequences in the plurality of the partially or fully ds DNA sequencing adapters is at least 100.
7 more elements
Dependencies are inferred from the claim text.