Abstract
Common bottlenecks in environmental and crop 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 Polymerase Chain Reaction (PCR) inhibitors. To address this, we have established a low‐cost genomic DNA extraction method for soil samples. We also present 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. (PLoS Biology, 15, e2003916, 2017). Our benchmarking experiment used four different soil types (coniferous, broad‐leafed, and mixed forest plus a standardized cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analyzing sequence variants of 16S V4 and ITS rRNA amplicons. We 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, for example, 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. Here, we present a low‐cost genomic DNA extraction method for soil samples 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, and Dovetail genomics.
Highlights
In the last decade, microbiome studies have been increasing rapidly in popularity, from 4505 publications by December 2010 to 66,250 publications by February 2020 (PubMed reports for search term “microbiome”)
We tested our SDE method by extracting genomic DNA (gDNA) from 250 mg samples of four different soil types taken from a mixed forest (MiF), a coniferous forest (CoF), and a broad-leafed forest (BrF), plus a standardized cereal crop compost mix used at the John Innes Centre (Cer)
The gDNA extracted with MP BiomedicalsTM FastDNATM SPIN, MoBio PowerSoil®, and our SDE method performed well in library construction, producing libraries with similar profiles (Figure 2)
Summary
Microbiome studies have been increasing rapidly in popularity, from 4505 publications by December 2010 to 66,250 publications by February 2020 (PubMed reports for search term “microbiome”). Commercial kits are limited to a small number of barcoded libraries (Minich, Humphrey, et al, 2018), while specialist workflows (e.g., the Earth Microbiome Project benchmarked protocols) use custom sequencing primers and cannot be processed using standard Illumina protocols. This limits the choice of the available sequencing provider and affects throughput and sequencing prices (Walters et al, 2016) restricting many projects to lower throughput in-house platforms such as the Illumina MiSeq. For comparison, the Illumina MiSeq v2 500cycle kit has an 8.5 Gb maximal output whereas the NovaSeq 6000 SP 500-cycle kit has a 400 Gb maximal output (Bahram et al, 2018; Bartoli et al, 2008; Thiergart et al, 2020). The combination of our two new protocols allows better utilization of state-of-the-art Illumina sequencing platforms to perform large-scale amplicon-based microbiome studies at a reduced cost
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