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

Karen Cravero, Arielle Medford, Aparna Pallavajjala, Jenna Canzoniero, Natasha Hunter, David Chu, Rory L. Cochran, Ian Waters, Eric S. Christenson, Kelly Kyker-Snowman, Berry Button, Alex J. Cole, Ben Ho Park

Research output: Contribution to journalArticlepeer-review

Abstract

Background: 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. Design and methods: 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. Results: 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. Conclusions: We demonstrate that BAmSeq allows original DNA template to be recovered with comparable quality and quantity to the source DNA. This recovered DNA is suitable for many downstream applications and may prevent sample exhaustion, especially when DNA quantity or source material is limiting.

Original languageEnglish (US)
Article numbere00108
JournalPractical Laboratory Medicine
Volume12
DOIs
StatePublished - Nov 2018

Keywords

  • Droplet digital PCR (ddPCR)
  • Next generation sequencing
  • Plasma DNA
  • Targeted amplicon sequencing

ASJC Scopus subject areas

  • Radiological and Ultrasound Technology
  • Clinical Biochemistry

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