Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Abstract

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

Human Activity Determines the Presence of Integron-Associated and Antibiotic Resistance Genes in Southwestern British Columbia

“…The bacterial fraction of each water sample was captured by passing water first through a 1 μm-Envirochek HV sampling capsules (Pall Corporation, Ann Harbor, MI, United States), followed by further filtration using a 0.22-μm 142 mm Supor-200 membrane disc filters (Pall Corporation, Ann Harbor, MI, United States) that retained bacterial cells as previously described ( Uyaguari-Diaz et al, 2015 , 2016 ). Filters were cut into small strips (1 cm × 1 cm) using sterile scissors and placed into 50 ml sterile centrifuge tubes (VWR, Radnor, PA, United States).…”

Characterization of Legionella Species from Watersheds in British Columbia, Canada

“…Amplicons were purified using the QIAQuick PCR purification kit (Qiagen Sciences, Maryland, MD), and sequencing libraries were prepared using the NEXTflex ChIP-Seq kit (BIOO Scientific, Austin, TX) using the gel size selection option, both per the manufacturer’s instructions. The shotgun sequencing libraries were prepared using the Nextera XT DNA sample preparation kit (Illumina, Inc., San Diego, CA), and gel-size selection was performed using the Ranger technology (Coastal Genomics Inc., Burnaby, BC, Canada) and targeting fragments between 500 and 800 bp ( 31 ).…”

Metagenomic Investigation of Plasma in Individuals with ME/CFS Highlights the Importance of Technical Controls to Elucidate Contamination and Batch Effects

“…Insert sizes of 300 to 400 bp were targeted in the size selection process incorporated in the NEBNext protocol, and an additional AMPure bead treatment performed to minimize the presence of adapter dimers. To further decrease the adapter dimer content in the final batch of 32 samples, a modified gel size selection approach targeting fragments between 300 and 500 bp using Ranger Technology (Coastal Genomics Inc., Burnaby, BC) was performed following library preparation [17]. …”