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Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing
Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extrem...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914102/ https://www.ncbi.nlm.nih.gov/pubmed/27060140 http://dx.doi.org/10.1093/nar/gkw224 |
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author | Ståhlberg, Anders Krzyzanowski, Paul M. Jackson, Jennifer B. Egyud, Matthew Stein, Lincoln Godfrey, Tony E. |
author_facet | Ståhlberg, Anders Krzyzanowski, Paul M. Jackson, Jennifer B. Egyud, Matthew Stein, Lincoln Godfrey, Tony E. |
author_sort | Ståhlberg, Anders |
collection | PubMed |
description | Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. |
format | Online Article Text |
id | pubmed-4914102 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-49141022016-06-22 Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing Ståhlberg, Anders Krzyzanowski, Paul M. Jackson, Jennifer B. Egyud, Matthew Stein, Lincoln Godfrey, Tony E. Nucleic Acids Res Methods Online Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. Oxford University Press 2016-06-20 2016-04-07 /pmc/articles/PMC4914102/ /pubmed/27060140 http://dx.doi.org/10.1093/nar/gkw224 Text en © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Ståhlberg, Anders Krzyzanowski, Paul M. Jackson, Jennifer B. Egyud, Matthew Stein, Lincoln Godfrey, Tony E. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing |
title | Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing |
title_full | Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing |
title_fullStr | Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing |
title_full_unstemmed | Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing |
title_short | Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing |
title_sort | simple, multiplexed, pcr-based barcoding of dna enables sensitive mutation detection in liquid biopsies using sequencing |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914102/ https://www.ncbi.nlm.nih.gov/pubmed/27060140 http://dx.doi.org/10.1093/nar/gkw224 |
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