Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity

Practical Laboratory Medicine
Karen CraveroBen Ho Park

Abstract

Genomic testing is often limited by the exhaustible nature of human tissue and blood samples. Here we describe biotinylated amplicon sequencing (BAmSeq), a method that allows for the creation of PCR amplicon based next-generation sequencing (NGS) libraries while retaining the original source DNA. Biotinylated primers for different loci were designed to create NGS libraries using human genomic DNA from cell lines, plasma, and formalin-fixed paraffin embedded (FFPE) tissues using the BAmSeq protocol. DNA from the original template used for each BAmSeq library was recovered after separation with streptavidin magnetic beads. The recovered DNA was then used for end-point, quantitative and droplet digital PCR (ddPCR) as well as NGS using a cancer gene panel. Recovered DNA was analyzed and compared to the original DNA after one or two rounds of BAmSeq. Recovered DNA revealed comparable genomic distributions and mutational allelic frequencies when compared to original source DNA. Sufficient quantities of recovered DNA after BAmSeq were obtained, allowing for additional downstream applications. We demonstrate that BAmSeq allows original DNA template to be recovered with comparable quality and quantity to the source DNA. This recovered D...Continue Reading

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Methods Mentioned

BETA
PCR
Amplicon Sequencing
pull down
Assay
biopsy
pull-down
BAmSeq

Software Mentioned

BWA aligner
Integrative Genomics Viewer
IGV
Bowtie
QuantaSoft
Picard Tools
COSMIC
Mutation Assessor Impact prediction
Integrated Genome Viewer
MDLVC

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