Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology

Abstract High-throughput sequencing platforms are increasingly being used for targeted amplicon sequencing because they enable cost-effective sequencing of large sample sets. For meaningful interpretation of targeted amplicon sequencing data and comparison between studies, it is critical that bioinf...

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Autores principales:	Yasemin Guenay-Greunke, David A. Bohan, Michael Traugott, Corinna Wallinger
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c0897562df0b401f821199e2ad00ba36
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spelling	oai:doaj.org-article:c0897562df0b401f821199e2ad00ba362021-12-02T17:18:21ZHandling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology10.1038/s41598-021-98018-42045-2322https://doaj.org/article/c0897562df0b401f821199e2ad00ba362021-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-98018-4https://doaj.org/toc/2045-2322Abstract High-throughput sequencing platforms are increasingly being used for targeted amplicon sequencing because they enable cost-effective sequencing of large sample sets. For meaningful interpretation of targeted amplicon sequencing data and comparison between studies, it is critical that bioinformatic analyses do not introduce artefacts and rely on detailed protocols to ensure that all methods are properly performed and documented. The analysis of large sample sets and the use of predefined indexes create challenges, such as adjusting the sequencing depth across samples and taking sequencing errors or index hopping into account. However, the potential biases these factors introduce to high-throughput amplicon sequencing data sets and how they may be overcome have rarely been addressed. On the example of a nested metabarcoding analysis of 1920 carabid beetle regurgitates to assess plant feeding, we investigated: (i) the variation in sequencing depth of individually tagged samples and the effect of library preparation on the data output; (ii) the influence of sequencing errors within index regions and its consequences for demultiplexing; and (iii) the effect of index hopping. Our results demonstrate that despite library quantification, large variation in read counts and sequencing depth occurred among samples and that the sequencing error rate in bioinformatic software is essential for accurate adapter/primer trimming and demultiplexing. Moreover, setting an index hopping threshold to avoid incorrect assignment of samples is highly recommended.Yasemin Guenay-GreunkeDavid A. BohanMichael TraugottCorinna WallingerNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yasemin Guenay-Greunke David A. Bohan Michael Traugott Corinna Wallinger Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology
description	Abstract High-throughput sequencing platforms are increasingly being used for targeted amplicon sequencing because they enable cost-effective sequencing of large sample sets. For meaningful interpretation of targeted amplicon sequencing data and comparison between studies, it is critical that bioinformatic analyses do not introduce artefacts and rely on detailed protocols to ensure that all methods are properly performed and documented. The analysis of large sample sets and the use of predefined indexes create challenges, such as adjusting the sequencing depth across samples and taking sequencing errors or index hopping into account. However, the potential biases these factors introduce to high-throughput amplicon sequencing data sets and how they may be overcome have rarely been addressed. On the example of a nested metabarcoding analysis of 1920 carabid beetle regurgitates to assess plant feeding, we investigated: (i) the variation in sequencing depth of individually tagged samples and the effect of library preparation on the data output; (ii) the influence of sequencing errors within index regions and its consequences for demultiplexing; and (iii) the effect of index hopping. Our results demonstrate that despite library quantification, large variation in read counts and sequencing depth occurred among samples and that the sequencing error rate in bioinformatic software is essential for accurate adapter/primer trimming and demultiplexing. Moreover, setting an index hopping threshold to avoid incorrect assignment of samples is highly recommended.
format	article
author	Yasemin Guenay-Greunke David A. Bohan Michael Traugott Corinna Wallinger
author_facet	Yasemin Guenay-Greunke David A. Bohan Michael Traugott Corinna Wallinger
author_sort	Yasemin Guenay-Greunke
title	Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology
title_short	Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology
title_full	Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology
title_fullStr	Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology
title_full_unstemmed	Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology
title_sort	handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/c0897562df0b401f821199e2ad00ba36
work_keys_str_mv	AT yaseminguenaygreunke handlingoftargetedampliconsequencingdatafocusingonindexhoppinganddemultiplexingusinganestedmetabarcodingapproachinecology AT davidabohan handlingoftargetedampliconsequencingdatafocusingonindexhoppinganddemultiplexingusinganestedmetabarcodingapproachinecology AT michaeltraugott handlingoftargetedampliconsequencingdatafocusingonindexhoppinganddemultiplexingusinganestedmetabarcodingapproachinecology AT corinnawallinger handlingoftargetedampliconsequencingdatafocusingonindexhoppinganddemultiplexingusinganestedmetabarcodingapproachinecology
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Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology

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