Automated Unbiased Metagenomic DNA Extraction for Long-Read Sequencing

Joshua Tjokrosurjo, Angel Gonzalez, Kevin Lin, Shuiquan Tang, Xi Yu Jia
2019 Journal of Biomolecular Techniques  
Long-read DNA sequencing is being touted as the next next-gen sequencing due to its affordability, ease of use, and increased output of more accurate data compared to traditional next-generation sequencing technologies. For example, the advantages of Nanopore long-read sequencing include the capacity to generate very long reads with remarkable speed and portability, spanning tandem-repeat regions, which resolves ambiguity during genome assembly. However, extracting inhibitor-free high molecular
more » ... weight (HMW) DNA suitable for long-read sequencing has always been a challenge due to DNA fragmentation during extraction caused by physical and enzymatic (DNases) breakage. Here we present an automated HMW DNA extraction pipeline that combines magnetic bead-based DNA extraction with the Microlab STAR liquid handler. Magnetic bead chemistry allows automated processing by retaining HMW DNA during stringent wash steps, leading to high quantities of long-read sequencing-ready DNA. To test the efficacy of this automated workflow, HMW DNA was isolated from a well-defined mixture of bacteria and yeast cells and subsequently sequenced using the MinION platform. Using this method, we achieved 1M reads, 8 Gb throughput, with average read-lengths of 8 kb, and over 100 kb recorded. Moreover, we find that the extracted microbial profile and proportional composition closely matches the theoretical composition. Overall, we have developed an automated method for HMW DNA extraction that shows unbiased microbial lysis that is compatible with long-read nanopore sequencing.
pmid:31892871 pmcid:PMC6936182 fatcat:re2o5uvdafgv7jcubexv6o5toi