High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing

Abstract

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput, comparatively to the actual sequencing costs. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS™ 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit excelled across all performance parameters. We further used the nanoliter dispensing system I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample. This enhanced efficiency and cost-effectiveness will enable researchers to undertake studies with greater sample sizes and diversity, thereby providing a richer, more detailed view of microbial communities and their dynamics.

Fig 1. Experimental design.

Top: Experimental design. Five different soil types were used to benchmark three different HT DNA extraction methods. DNA extractions were evaluated by DNA quality, quantity, length, and observed community profile. The I.DOT One was subsequently used to miniaturize metagenomes and amplicons. Bottom: Hands-on time, total time, and cost associated with each step from DNA extraction to prepared metagenome or amplicon libraries. The time reflects the processing time of a full 96-well plate, whereas costs are calculated per sample. For both sequencing strategies, two 96-well plates can be processed concurrently with only a minor increase in total time.

Fig 2. Community characteristics for DNA extraction kits.

(A) Heatmap of community profile at phylum level across DNA extraction kits faceted by soil type. (B) PCA on Hellinger transformed relative abundance. (C) PCA on Hellinger transformed relative abundance for the Clay samples only. For all plots: ASVs not exceeding 0.1% relative abundance in at least one sample were filtered out.

Fig 3. Comparison of community characteristics between standard and miniaturized amplicons.

(A) Heatmap of community profile at the genus level across reaction volume faceted by soil type. (B) Differential relative abundance plot of ASVs. (C) Hellinger transformed relative abundance, PCA. ASVs not exceeding 0.1% relative abundance in at least one sample were filtered out.

Fig 4

(A) Heatmap of community profile at the genus level across reaction volume faceted by soil type. (B) Differential abundance plot of all genera (C) Hellinger transformed relative abundance, PCA. Genera which did not exceed 0.1% relative abundance in at least one sample were filtered out.