Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity
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 so...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2018-11-01
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| Series: | Practical Laboratory Medicine |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352551718300507 |
| _version_ | 1811268471293476864 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Karen Cravero |
| collection | DOAJ |
| description | 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. Keywords: Next generation sequencing, Plasma DNA, Droplet digital PCR (ddPCR), Targeted amplicon sequencing |
| first_indexed | 2024-04-12T21:23:29Z |
| format | Article |
| id | doaj.art-3e2495c1fac645eb88e783d29a514742 |
| institution | Directory Open Access Journal |
| issn | 2352-5517 |
| language | English |
| last_indexed | 2024-04-12T21:23:29Z |
| publishDate | 2018-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Practical Laboratory Medicine |
| spelling | doaj.art-3e2495c1fac645eb88e783d29a5147422022-12-22T03:16:15ZengElsevierPractical Laboratory Medicine2352-55172018-11-0112Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantityKaren Cravero0Arielle Medford1Aparna Pallavajjala2Jenna Canzoniero3Natasha Hunter4David Chu5Rory L. Cochran6Ian Waters7Eric S. Christenson8Kelly Kyker-Snowman9Berry Button10Alex J. Cole11Ben Ho Park12The Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesDepartment of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United StatesThe Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States; The Whiting School of Engineering, Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, United States; Correspondence to: 1650 Orleans Street, Room 151, Baltimore, MD 21287, United States.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. Keywords: Next generation sequencing, Plasma DNA, Droplet digital PCR (ddPCR), Targeted amplicon sequencinghttp://www.sciencedirect.com/science/article/pii/S2352551718300507 |
| spellingShingle | 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 Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity Practical Laboratory Medicine |
| title | Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity |
| title_full | Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity |
| title_fullStr | Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity |
| title_full_unstemmed | Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity |
| title_short | Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity |
| title_sort | biotinylated amplicon sequencing a method for preserving dna samples of limited quantity |
| url | http://www.sciencedirect.com/science/article/pii/S2352551718300507 |
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