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US 2022/0033901 A1Published application
Universal sanger sequencing from next-gen sequencing amplicons
AssigneeQuest Diagnostics Investments Incorporated
InventorsHeather Sanders, Hai-Rong Li, Feras Hantash, Frederic Waldman
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Abstract
Described herein are methods, compositions and kits directed to amplification of nucleic acids suitable for both next generation sequencing (NGS) and a second round of sequencing as validation, such as Sanger sequencing.
Claims10
-20. (canceled)
A method of generating an amplicon for next generation sequencing (NSG) for at least one region of interest comprising:
- (a) adding to a sample of DNA:
- (i) a first primer set specific for the region of interest consisting of two primers each comprising a 5′ region encoding an adapter and a 3′ region specific for the region of interest;
- and
- (ii) a second primer set consisting of two primers each of which comprises:
- (A) a 5′ sequence selected from GTAAAACGACGGCCAGT (SEQ ID NO:
- 1) or AACAGCTATGACCATG (SEQ ID NO:
- 2);
- (B) a spacer comprising at least 10 nucleotides;
- and
- (C) a 3′ adapter that is substantially identical to the adaptor region,
- wherein the adapter has a Tm of about 55° C. and the second primer set has a Tm of at least 70° C.;
- and
- (b) performing a plurality of PCR cycles with a first annealing temperature and a second annealing temperature.
10 more elements
Dependencies are inferred from the claim text.