A low‐cost pipeline for soil microbiome profiling. Issue 12 (22nd November 2020)

• Record Type: Journal Article
• Authors: Bollmann‐Giolai, Anita; Giolai, Michael; Heavens, Darren; Macaulay, Iain; Malone, Jacob; Clark, Matthew D.
• Is Part Of: MicrobiologyOpen. Volume 9:Issue 12(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-11-22
• Subjects: amplicon library construction -- DNA cleanup -- gDNA extraction -- microbiome -- soil; Microbiology -- Periodicals; 579
• DOI: 10.1002/mbo3.1133
• Languages: English
• ISSNs: 2045-8827

Abstract

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. Abstract : We present an inexpensive and high‐throughput magnetic particle‐based soil genomic DNA (gDNA) extraction method "SDE" ($0.36) for next‐generation sequencing quality gDNA extraction, scalable to thousands of samples with little hands‐on time. SDE can also be used in combination with our low‐cost amplicon library construction workflow (16S, ITS, $2.5), enabling multiplexing of up to 2304 libraries at a time. Combining SDE and our custom library construction workflow allows utilization of state‐of‐the‐art Illumina sequencing platforms (e.g., NovaSeq) for large‐scale microbiome studies.