Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples.
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.
Department of Pathology and Laboratory Medicine, Faculty of Medicine, The University of British Columbia; Miguel.UyaguariDiaz@bccdc.ca.
Miguel I
MI
Uyaguari-Diaz
Coastal Genomics.
Jared R
JR
Slobodan
Coastal Genomics.
Matthew J
MJ
Nesbitt
British Columbia Public Health Microbiology and Reference Laboratory.
Matthew A
MA
Croxen
Department of Pathology and Laboratory Medicine, Faculty of Medicine, The University of British Columbia; British Columbia Public Health Microbiology and Reference Laboratory.
Judith
J
Isaac-Renton
Department of Pathology and Laboratory Medicine, Faculty of Medicine, The University of British Columbia; British Columbia Public Health Microbiology and Reference Laboratory.
Natalie A
NA
Prystajecky
Department of Pathology and Laboratory Medicine, Faculty of Medicine, The University of British Columbia; British Columbia Public Health Microbiology and Reference Laboratory.
Patrick
P
Tang
D001331:Automation; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004586:Electrophoresis / Q000379:methods*; D004777:Environment; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D017422:Sequence Analysis, DNA / Q000379:methods; D014874:Water Pollutants, Chemical / Q000737:chemistry* / Q000302:isolation & purification
D014874:Water Pollutants, Chemical; D004247:DNA