High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

ABSTRACT Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods h...

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Autores principales:	Jeremiah J. Minich, Greg Humphrey, Rodolfo A. S. Benitez, Jon Sanders, Austin Swafford, Eric E. Allen, Rob Knight
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	DNA metabarcoding Illumina MiSeq NGS acoustic liquid handler automation library preparation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/dce366e8d965490e8089c12c3f2d80ab
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spelling	oai:doaj.org-article:dce366e8d965490e8089c12c3f2d80ab2021-12-02T19:47:34ZHigh-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity10.1128/mSystems.00166-182379-5077https://doaj.org/article/dce366e8d965490e8089c12c3f2d80ab2018-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00166-18https://doaj.org/toc/2379-5077ABSTRACT Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (R2 > 0.95), while distances of technical replicates were much lower than within-body-site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements. IMPORTANCE Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.Jeremiah J. MinichGreg HumphreyRodolfo A. S. BenitezJon SandersAustin SwaffordEric E. AllenRob KnightAmerican Society for MicrobiologyarticleDNA metabarcodingIllumina MiSeqNGSacoustic liquid handlerautomationlibrary preparationMicrobiologyQR1-502ENmSystems, Vol 3, Iss 6 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	DNA metabarcoding Illumina MiSeq NGS acoustic liquid handler automation library preparation Microbiology QR1-502
spellingShingle	DNA metabarcoding Illumina MiSeq NGS acoustic liquid handler automation library preparation Microbiology QR1-502 Jeremiah J. Minich Greg Humphrey Rodolfo A. S. Benitez Jon Sanders Austin Swafford Eric E. Allen Rob Knight High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
description	ABSTRACT Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (R2 > 0.95), while distances of technical replicates were much lower than within-body-site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements. IMPORTANCE Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.
format	article
author	Jeremiah J. Minich Greg Humphrey Rodolfo A. S. Benitez Jon Sanders Austin Swafford Eric E. Allen Rob Knight
author_facet	Jeremiah J. Minich Greg Humphrey Rodolfo A. S. Benitez Jon Sanders Austin Swafford Eric E. Allen Rob Knight
author_sort	Jeremiah J. Minich
title	High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
title_short	High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
title_full	High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
title_fullStr	High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
title_full_unstemmed	High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity
title_sort	high-throughput miniaturized 16s rrna amplicon library preparation reduces costs while preserving microbiome integrity
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/dce366e8d965490e8089c12c3f2d80ab
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High-Throughput Miniaturized 16S rRNA Amplicon Library Preparation Reduces Costs while Preserving Microbiome Integrity

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