Barcode-free next-generation sequencing error validation for ultra-rare variant detection

Nature Communications
Huiran YeomSunghoon Kwon

Abstract

The advent of next-generation sequencing (NGS) has accelerated biomedical research by enabling the high-throughput analysis of DNA sequences at a very low cost. However, NGS has limitations in detecting rare-frequency variants (< 1%) because of high sequencing errors (> 0.1~1%). NGS errors could be filtered out using molecular barcodes, by comparing read replicates among those with the same barcodes. Accordingly, these barcoding methods require redundant reads of non-target sequences, resulting in high sequencing cost. Here, we present a cost-effective NGS error validation method in a barcode-free manner. By physically extracting and individually amplifying the DNA clones of erroneous reads, we distinguish true variants of frequency > 0.003% from the systematic NGS error and selectively validate NGS error after NGS. We achieve a PCR-induced error rate of 2.5×10-6 per base per doubling event, using 10 times less sequencing reads compared to those from previous studies.

References

Jul 31, 2010·Nucleic Acids Research·Osvaldo ZagordiNiko Beerenwinkel
Nov 10, 2010·Nucleic Acids Research·H JohanssonMats Nilsson
Mar 15, 2011·Nature·Nicholas NavinMichael Wigler
May 19, 2011·Proceedings of the National Academy of Sciences of the United States of America·Isaac KindeBert Vogelstein
Nov 22, 2011·Nature Methods·Teemu KiviojaJussi Taipale
Dec 20, 2011·Nature Biotechnology·Joke ReumersJurgen Del-Favero
Jan 11, 2012·Proceedings of the National Academy of Sciences of the United States of America·Katsuyuki ShiroguchiX Sunney Xie
Jul 19, 2012·European Journal of Immunology·Dmitry A BolotinDmitriy M Chudakov
Aug 3, 2012·Proceedings of the National Academy of Sciences of the United States of America·Michael W SchmittLawrence A Loeb
Jan 17, 2014·Nature Communications·Niccolo BolliNikhil C Munshi
May 6, 2014·Nature Methods·Mikhail ShugayDmitriy M Chudakov
Apr 8, 2015·Nature Medicine·Hyo-eun C BhangFrank Stegmeier
Apr 17, 2015·Science Translational Medicine·Siân JonesVictor E Velculescu
May 31, 2015·Briefings in Bioinformatics·David LaehnemannAlice Carolyn McHardy
Feb 2, 2016·Mutation Research·Matthew S HestandJoris R Vermeesch
May 18, 2016·Nature Reviews. Genetics·Sara GoodwinW Richard McCombie
Sep 27, 2016·Applied & Translational Genomics·Christoph EndrullatMarcus Frohme
Jan 7, 2017·PloS One·Vladimir Potapov, Jennifer L Ong
Jan 28, 2017·Scientific Reports·Michiru NishitaYasuhiro Minami

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Methods Mentioned

BETA
PCR
Illumina sequencing
454 sequencing
chip

Software Mentioned

VasrScan
- toolkit
FASTX
NGS
BLAST Aligner
LabVIEW
BLAST

Related Concepts

Related Feeds

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.