Dataset Information

NanoGBS: A Miniaturized Procedure for GBS Library Preparation.

ABSTRACT: High-throughput reduced-representation sequencing (RRS)-based genotyping methods, such as genotyping-by-sequencing (GBS), have provided attractive genotyping solutions in numerous species. Here, we present NanoGBS, a miniaturized and eco-friendly method for GBS library construction. Using acoustic droplet ejection (ADE) technology, NanoGBS libraries were constructed in tenfold smaller volumes compared to standard methods (StdGBS) and leading to a reduced use of plastics of up to 90%. A high-quality DNA library and SNP catalogue were obtained with extensive overlap (96%) in SNP loci and 100% agreement in genotype calls compared to the StdGBS dataset with a high level of accuracy (98.5%). A highly multiplexed pool of GBS libraries (768-plex) was sequenced on a single Ion Proton PI chip and yielded enough SNPs (~4K SNPs; 1.5 SNP per cM, on average) for many high-volume applications. Combining NanoGBS library preparation and increased multiplexing can dramatically reduce (72%) genotyping cost per sample. We believe that this approach will greatly facilitate the adoption of marker applications where extremely high throughputs are required and cost is still currently limiting.

SUBMITTER: Torkamaneh D

PROVIDER: S-EPMC7040475 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

NanoGBS: A Miniaturized Procedure for GBS Library Preparation.

Torkamaneh Davoud D Boyle Brian B St-Cyr Jérôme J Légaré Gaétan G Pomerleau Sonia S Belzile François F

Frontiers in genetics 20200218

High-throughput reduced-representation sequencing (RRS)-based genotyping methods, such as genotyping-by-sequencing (GBS), have provided attractive genotyping solutions in numerous species. Here, we present NanoGBS, a miniaturized and eco-friendly method for GBS library construction. Using acoustic droplet ejection (ADE) technology, NanoGBS libraries were constructed in tenfold smaller volumes compared to standard methods (StdGBS) and leading to a reduced use of plastics of up to 90%. A high-qual ...[more]

PMID: 32133028

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Project description: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 (R 2 > 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.

Dataset Information

NanoGBS: A Miniaturized Procedure for GBS Library Preparation.

Publications

NanoGBS: A Miniaturized Procedure for GBS Library Preparation.

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