Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding

Environmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples and is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding. The Illumina iSeq 100 (hereafter, iSeq), one of the high-throughput sequencers (HTS), has a relatively simple workflow and is potentially more affordable than other HTS. However, its utility in eDNA metabarcoding has still not been investigated. In the present study, we applied fish eDNA metabarcoding to 40 water samples from river and lake ecosystems to assess the difference in species detectability and composition between iSeq and MiSeq. To check differences in sequence quality and errors, we also assessed differences in read changes between the two HTS. There were similar sequence qualities between iSeq and MiSeq. Significant difference was observed in the number of species between two HTS, but no difference was observed in species composition between the two HTS. Additionally, the species compositions in common with the conventional method were the same between the two HTS. According to the results, using the same amplicon library for sequencing, two HTS would exhibit a similar performance of fish species detection using eDNA metabarcoding.

Microbial map of the world’s vineyards: Applying the concept of microbial terroir on a global scale

The specific microbial biodiversity linked to a particular vineyard location is reported to be a crucial aspect, in conjunction with edaphic, climatic and human factors, in the concept of wine terroir. These biogeographical patterns are known as microbial terroirs.This study applied an HTS amplicon library approach in order to conduct a global survey of vineyards’ soil microbial communities. In all, soil samples from 200 vineyards on four continents were analysed in an attempt to establish the basis for the development of a vineyard soil microbiome map to represent microbial wine terroirs on a global scale.This study established links between vineyard locations and microbial biodiversity on different scales: between continents and countries, and between different wine regions within the same country. Geography had a strong effect on the composition of microbial communities on a global scale, which was also maintained on a country scale. Furthermore, a predictive model was developed, based on random forest analyses, to discriminate between microbial patterns in order to identify the geographical source of the samples with reasonable precision. Finally this study is the first to describe the microbial community of new and northern wine-producing regions, such as Denmark, that could be of great interest for viticulture adaptation in a context of climate change.

Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding

Environmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples, and it is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding, one of the eDNA analysis approaches. The Illumina iSeq 100 (hereafter, iSeq) is one of the numerous high-throughput sequencers (HTS). It has a relatively simple workflow and is potentially more affordable than other sequencers for deployment in HTS environments. However, to date, only a few studies have adopted iSeq, and its utility in eDNA metabarcoding has still not been investigated comprehensively. In the present study, we applied fish eDNA metabarcoding to river and lake environmental samples using iSeq and MiSeq approaches. We also assessed differences in fish species detectability among iSeq, MiSeq, and conventional approaches. Twenty-seven river and 13 lake samples were amplified using MiFish primers and sequenced with iSeq and MiSeq, respectively. The iSeq and MiSeq metabarcoding achieved high detectability for fish taxa in the ecosystems. Species numbers and compositions in each river detected using iSeq were almost consistent with those of MiSeq, indicating detectability of both techniques was comparable. The comparison of the species compositions of the two HTSs with those of conventional methods showed that the common species between each HTS and the conventional methods were exactly similar. According to the results, if the same amplicon library were used for sequencing, there would be negligible detectability differences between iSeq and MiSeq based on eDNA metabarcoding.

A low-cost pipeline for soil microbiome profiling

BackgroundCommon bottlenecks in environmental microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in PCR inhibitors. To address this, we have established a low-cost genomic DNA extraction method for inhibitor rich samples alongside an Illumina-compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non-commercial extraction method by Zou et al. (2017). Our benchmarking experiment used four different soil types (coniferous, broad leafed, and mixed forest plus a standardised cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analysing sequence variants of 16S V4 and ITS rRNA amplicons.ResultsWe found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column-based DNA extraction with extensive washing delivered the cleanest genomic DNA e.g. best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits.ConclusionHere we present a low-cost genomic DNA extraction method for inhibitor rich sample types such as soil that can be coupled to an Illumina-compatible simple two step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high quality genomic DNA at a fraction of the cost of commercial kits and enables cost-effective, large scale amplicon sequencing projects. Notably our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, Dovetail genomics etc.

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 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.