Triplicate PCR reactions for 16S rRNA gene amplicon sequencing are unnecessary

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Abstract

Conventional wisdom holds that PCR amplification for sequencing should employ pooled replicate reactions to reduce bias due to jackpot effects and chimera formation. However, modern amplicon data analysis employs methods that may be less sensitive to such artifacts. Here we directly compare results from single versus triplicate reactions for 16S amplicon sequencing and find no significant impact of adopting a less labor-intensive single-reaction protocol.

Abstract

We compared single PCR reactions to pooled triplicate PCR reactions for 16S rRNA gene amplicon sequencing on nearly 400 samples from a diverse range of environments across three independent laboratories.

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Bias in template-to-product ratios in multitemplate PCR.

Martin F. Polz, Colleen Cavanaugh (1998)

Bias introduced by the simultaneous amplification of specific genes from complex mixtures of templates remains poorly understood. To explore potential causes and the extent of bias in PCR amplification of 16S ribosomal DNAs (rDNAs), genomic DNAs of two closely and one distantly related bacterial species were mixed and amplified with universal, degenerate primers. Quantification and comparison of template and product ratios showed that there was considerable and reproducible overamplification of specific templates. Variability between replicates also contributed to the observed bias but in a comparatively minor way. Based on these initial observations, template dosage and differences in binding energies of permutations of the degenerate, universal primers were tested as two likely causes of this template-specific bias by using 16S rDNA templates modified by site-directed mutagenesis. When mixtures of mutagenized templates containing AT- and GC-rich priming sites were used, templates containing the GC-rich permutation amplified with higher efficiency, indicating that different primer binding energies may to a large extent be responsible for overamplification. In contrast, gene copy number was found to be an unlikely cause of the observed bias. Similarly, amplification from DNA extracted from a natural community to which different amounts of genomic DNA of a single bacterial species were added did not affect relative product ratios. Bias was reduced considerably by using high template concentrations, by performing fewer cycles, and by mixing replicate reaction preparations.

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Sequence Depth, Not PCR Replication, Improves Ecological Inference from Next Generation DNA Sequencing

Dylan P Smith, Kabir G Peay (2014)

Recent advances in molecular approaches and DNA sequencing have greatly progressed the field of ecology and allowed for the study of complex communities in unprecedented detail. Next generation sequencing (NGS) can reveal powerful insights into the diversity, composition, and dynamics of cryptic organisms, but results may be sensitive to a number of technical factors, including molecular practices used to generate amplicons, sequencing technology, and data processing. Despite the popularity of some techniques over others, explicit tests of the relative benefits they convey in molecular ecology studies remain scarce. Here we tested the effects of PCR replication, sequencing depth, and sequencing platform on ecological inference drawn from environmental samples of soil fungi. We sequenced replicates of three soil samples taken from pine biomes in North America represented by pools of either one, two, four, eight, or sixteen PCR replicates with both 454 pyrosequencing and Illumina MiSeq. Increasing the number of pooled PCR replicates had no detectable effect on measures of α- and β-diversity. Pseudo-β-diversity – which we define as dissimilarity between re-sequenced replicates of the same sample – decreased markedly with increasing sampling depth. The total richness recovered with Illumina was significantly higher than with 454, but measures of α- and β-diversity between a larger set of fungal samples sequenced on both platforms were highly correlated. Our results suggest that molecular ecology studies will benefit more from investing in robust sequencing technologies than from replicating PCRs. This study also demonstrates the potential for continuous integration of older datasets with newer technology.

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Studying the microbiology of the indoor environment

Scott Kelley, Jack A Gilbert (2013)

The majority of people in the developed world spend more than 90% of their lives indoors. Here, we examine our understanding of the bacteria that co-inhabit our artificial world and how they might influence human health.

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Author and article information

Journal

Journal ID (publisher-id): BTN

Title: BioTechniques

Publisher: Future Science Ltd (London, UK )

ISSN (Print): 0736-6205

ISSN (Electronic): 1940-9818

Publication date (Electronic, pub): 24 May 2019

Publication date (Print, pub): July 2019

Volume: 67

Issue: 1

Pages: 29-32

Affiliations

¹Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA

²Division of Bioscience, Argonne National Laboratory University of Chicago, Chicago, IL, USA

³Department of Energy Joint Genome Institute, Walnut Creek, CA, USA

⁵Center for Microbiome Innovation, University of California San Diego, La Jolla, CA, USA

⁶Department of Computer Science and Engineering, University of California San Diego, La Jolla, CA, USA

Author notes

[* ]Author for correspondence: robknight@ 123456ucsd.edu

Article

DOI: 10.2144/btn-2018-0192

PMC ID: 7030937

PubMed ID: 31124709

SO-VID: b21e08a0-bafd-46d6-ba56-307d0e25ed25

License:

This work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License

History

Date received : 20 December 2018

Date accepted : 18 April 2019

Date: 24 May 2019

Page count

Pages: 4

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Triplicate PCR reactions for 16S rRNA gene amplicon sequencing are unnecessary

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Abstract

Abstract

Most cited references 7

Bias in template-to-product ratios in multitemplate PCR.

Sequence Depth, Not PCR Replication, Improves Ecological Inference from Next Generation DNA Sequencing

Studying the microbiology of the indoor environment

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Cited by 27

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