TY - JOUR
T1 - Biotinylated amplicon sequencing
T2 - A method for preserving DNA samples of limited quantity
AU - Cravero, Karen
AU - Medford, Arielle
AU - Pallavajjala, Aparna
AU - Canzoniero, Jenna
AU - Hunter, Natasha
AU - Chu, David
AU - Cochran, Rory L.
AU - Waters, Ian
AU - Christenson, Eric S.
AU - Kyker-Snowman, Kelly
AU - Button, Berry
AU - Cole, Alex J.
AU - Park, Ben Ho
N1 - Funding Information:
This work was supported by: The Komen Foundation (B.H.P.) , NIH CA088843 and CA194024 (K.C. and B.H.P.), We would also like to thank and acknowledge the support of the Sandy Garcia Charitable Foundation , the Commonwealth Foundation , the Santa Fe Foundation , the Breast Cancer Research Foundation , and the ME Foundation . None of the funding sources influenced the design, interpretation or submission of this manuscript.
Publisher Copyright:
© 2018 The Authors
PY - 2018/11
Y1 - 2018/11
N2 - 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.
AB - 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.
KW - Droplet digital PCR (ddPCR)
KW - Next generation sequencing
KW - Plasma DNA
KW - Targeted amplicon sequencing
UR - http://www.scopus.com/inward/record.url?scp=85051663304&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85051663304&partnerID=8YFLogxK
U2 - 10.1016/j.plabm.2018.e00108
DO - 10.1016/j.plabm.2018.e00108
M3 - Article
C2 - 30140723
AN - SCOPUS:85051663304
SN - 2352-5517
VL - 12
JO - Practical Laboratory Medicine
JF - Practical Laboratory Medicine
M1 - e00108
ER -