Taxon Disappearance from Microbiome Analysis Reinforces the Value of Mock Communities as a Standard in Every Sequencing Run

ABSTRACT Mock communities have been used in microbiome method development to help estimate biases introduced in PCR amplification and sequencing and to optimize pipeline outputs. Nevertheless, the strong value of routine mock community analysis beyond initial method development is rarely, if ever, c...

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Autores principales:	Yi-Chun Yeh, David M. Needham, Ella T. Sieradzki, Jed A. Fuhrman
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	DNA sequencing microbiome analysis mock community Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b0532824a33b4758bd80d611a3335e44
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spelling	oai:doaj.org-article:b0532824a33b4758bd80d611a3335e442021-12-02T18:15:44ZTaxon Disappearance from Microbiome Analysis Reinforces the Value of Mock Communities as a Standard in Every Sequencing Run10.1128/mSystems.00023-182379-5077https://doaj.org/article/b0532824a33b4758bd80d611a3335e442018-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00023-18https://doaj.org/toc/2379-5077ABSTRACT Mock communities have been used in microbiome method development to help estimate biases introduced in PCR amplification and sequencing and to optimize pipeline outputs. Nevertheless, the strong value of routine mock community analysis beyond initial method development is rarely, if ever, considered. Here we report that our routine use of mock communities as internal standards allowed us to discover highly aberrant and strong biases in the relative proportions of multiple taxa in a single Illumina HiSeqPE250 run. In this run, an important archaeal taxon virtually disappeared from all samples, and other mock community taxa showed >2-fold high or low abundance, whereas a rerun of those identical amplicons (from the same reaction tubes) on a different date yielded “normal” results. Although obvious from the strange mock community results, we could have easily missed the problem had we not used the mock communities because of natural variation of microbiomes at our site. The “normal” results were validated over four MiSeqPE300 runs and three HiSeqPE250 runs, and run-to-run variation was usually low. While validating these “normal” results, we also discovered that some mock microbial taxa had relatively modest, but consistent, differences between sequencing platforms. We strongly advise the use of mock communities in every sequencing run to distinguish potentially serious aberrations from natural variations. The mock communities should have more than just a few members and ideally at least partly represent the samples being analyzed to detect problems that show up only in some taxa and also to help validate clustering. IMPORTANCE Despite the routine use of standards and blanks in virtually all chemical or physical assays and most biological studies (a kind of “control”), microbiome analysis has traditionally lacked such standards. Here we show that unexpected problems of unknown origin can occur in such sequencing runs and yield completely incorrect results that would not necessarily be detected without the use of standards. Assuming that the microbiome sequencing analysis works properly every time risks serious errors that can be detected by the use of mock communities.Yi-Chun YehDavid M. NeedhamElla T. SieradzkiJed A. FuhrmanAmerican Society for MicrobiologyarticleDNA sequencingmicrobiome analysismock communityMicrobiologyQR1-502ENmSystems, Vol 3, Iss 3 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	DNA sequencing microbiome analysis mock community Microbiology QR1-502
spellingShingle	DNA sequencing microbiome analysis mock community Microbiology QR1-502 Yi-Chun Yeh David M. Needham Ella T. Sieradzki Jed A. Fuhrman Taxon Disappearance from Microbiome Analysis Reinforces the Value of Mock Communities as a Standard in Every Sequencing Run
description	ABSTRACT Mock communities have been used in microbiome method development to help estimate biases introduced in PCR amplification and sequencing and to optimize pipeline outputs. Nevertheless, the strong value of routine mock community analysis beyond initial method development is rarely, if ever, considered. Here we report that our routine use of mock communities as internal standards allowed us to discover highly aberrant and strong biases in the relative proportions of multiple taxa in a single Illumina HiSeqPE250 run. In this run, an important archaeal taxon virtually disappeared from all samples, and other mock community taxa showed >2-fold high or low abundance, whereas a rerun of those identical amplicons (from the same reaction tubes) on a different date yielded “normal” results. Although obvious from the strange mock community results, we could have easily missed the problem had we not used the mock communities because of natural variation of microbiomes at our site. The “normal” results were validated over four MiSeqPE300 runs and three HiSeqPE250 runs, and run-to-run variation was usually low. While validating these “normal” results, we also discovered that some mock microbial taxa had relatively modest, but consistent, differences between sequencing platforms. We strongly advise the use of mock communities in every sequencing run to distinguish potentially serious aberrations from natural variations. The mock communities should have more than just a few members and ideally at least partly represent the samples being analyzed to detect problems that show up only in some taxa and also to help validate clustering. IMPORTANCE Despite the routine use of standards and blanks in virtually all chemical or physical assays and most biological studies (a kind of “control”), microbiome analysis has traditionally lacked such standards. Here we show that unexpected problems of unknown origin can occur in such sequencing runs and yield completely incorrect results that would not necessarily be detected without the use of standards. Assuming that the microbiome sequencing analysis works properly every time risks serious errors that can be detected by the use of mock communities.
format	article
author	Yi-Chun Yeh David M. Needham Ella T. Sieradzki Jed A. Fuhrman
author_facet	Yi-Chun Yeh David M. Needham Ella T. Sieradzki Jed A. Fuhrman
author_sort	Yi-Chun Yeh
title	Taxon Disappearance from Microbiome Analysis Reinforces the Value of Mock Communities as a Standard in Every Sequencing Run
title_short	Taxon Disappearance from Microbiome Analysis Reinforces the Value of Mock Communities as a Standard in Every Sequencing Run
title_full	Taxon Disappearance from Microbiome Analysis Reinforces the Value of Mock Communities as a Standard in Every Sequencing Run
title_fullStr	Taxon Disappearance from Microbiome Analysis Reinforces the Value of Mock Communities as a Standard in Every Sequencing Run
title_full_unstemmed	Taxon Disappearance from Microbiome Analysis Reinforces the Value of Mock Communities as a Standard in Every Sequencing Run
title_sort	taxon disappearance from microbiome analysis reinforces the value of mock communities as a standard in every sequencing run
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/b0532824a33b4758bd80d611a3335e44
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Taxon Disappearance from Microbiome Analysis Reinforces the Value of Mock Communities as a Standard in Every Sequencing Run

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