UNIVERSAL SANGER SEQUENCING FROM NEXT-GEN SEQUENCING AMPLICONS

摘要

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.

主权项

1. A composition comprising a first oligonucleotide that comprises, in 5′ to 3′ order,
(a) a first region suitable for use as a Sanger sequencing primer; (b) a spacer that is between about 5 and about 15 nucleotides long; and (c) a third region suitable for use as a next generation sequencing (NGS) primer, and a second oligonucleotide that comprises, in 5′ to 3′ order, (d) a first region that is substantially identical to the third region of the first oligonucleotide; and (e) a second region that is suitable for use as a polymerase chain reaction (PCR) primer, wherein the third region of the first oligonucleotide and the first region of the second oligonucleotide have a melting temperature (Tm) that is at least about 5° C. higher than the Tm of the second region of the second oligonucleotide, and wherein the total length of the spacer and the first and second regions of the second oligonucleotide is at least about 45 nucleotides (nt) long.