Systematic evaluation of error rates and causes in short samples in next-generation sequencing

Scientific Reports
Franziska PfeifferGünter Mayer

Abstract

Next-generation sequencing (NGS) is the method of choice when large numbers of sequences have to be obtained. While the technique is widely applied, varying error rates have been observed. We analysed millions of reads obtained after sequencing of one single sequence on an Illumina sequencer. According to our analysis, the index-PCR for sample preparation has no effect on the observed error rate, even though PCR is traditionally seen as one of the major contributors to enhanced error rates in NGS. In addition, we observed very persistent pre-phasing effects although the base calling software corrects for these. Removal of shortened sequences abolished these effects and allowed analysis of the actual mutations. The average error rate determined was 0.24 ± 0.06% per base and the percentage of mutated sequences was found to be 6.4 ± 1.24%. Constant regions at the 5'- and 3'-end, e.g., primer binding sites used in in vitro selection procedures seem to have no effect on mutation rates and re-sequencing of samples obtains very reproducible results. As phasing effects and other sequencing problems vary between equipment and individual setups, we recommend evaluation of error rates and types to all NGS-users to improve the quality and ...Continue Reading

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Citations

Oct 26, 2018·Human Mutation·Francesc MuyasStephan Ossowski
Jan 3, 2019·Genomics & Informatics·TaeJin AhnHyunsoo Park
May 10, 2019·Scientific Reports·Anna L McNaughtonPhilippa C Matthews
Jul 4, 2019·Nucleic Acids Research·Benjamin J CallahanMichael K Dougherty
Jul 19, 2019·Critical Reviews in Clinical Laboratory Sciences·Lize CuypersMahmoud Reza Pourkarim
Nov 12, 2019·Ecological Applications : a Publication of the Ecological Society of America·Xavier TuronOwen Simon Wangensteen
Aug 15, 2019·Journal of Human Genetics·Ahmed N AlkanaqNaomichi Matsumoto
May 5, 2020·NAR Genomics and Bioinformatics·Tiffany M DelhommeMatthieu Foll
Dec 18, 2019·BMC Bioinformatics·Anna-Katharina LauBurkhard Morgenstern
Apr 11, 2020·Ecology and Evolution·Elena M Duke, Ronald S Burton
Oct 23, 2019·Scientific Reports·Irena Fischer-HwangMikel Hernaez
Feb 26, 2020·Nature Ecology & Evolution·Weronika JasinskaShimon Bershtein
Aug 8, 2020·Ecology and Evolution·Shi-Yi ChenWei Wang
Mar 10, 2020·PeerJ·Dana L CarperDavid J Weston
Nov 6, 2020·Nucleic Acids Research·Iana FedorovaKonstantin Severinov
Jan 17, 2021·Virology Journal·Ryan V MoriartyShelby L O'Connor
Sep 10, 2020·IScience·Lucia CsepregiSai T Reddy
Feb 16, 2021·Frontiers in Microbiology·Muhammad Shafiq ShahidAbdullah M Al-Sadi
Feb 21, 2021·Genome Biology·Stephan Schmeing, Mark D Robinson
Feb 23, 2021·Molecular Ecology·Tyson H Holmes

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Methods Mentioned

BETA
exponential enrichment
SELEX
electrophoresis
PCR

Software Mentioned

COMPAS
Bustard
bcl2fastq2
AYB
MiSeq

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