lcWGS of Sardinops sagax : MTC101422B_A01
Genomic DNA was extracted from muscle tissue or fin clips stored in 100% ethanol using Qiagen DNAeasy Blood & Tissue 96 extraction kits (Qiagen, Inc., Valencia, CA). Extractions were run on a standard 2% agarose gel to screen for high molecular weight DNA and were then quantified using a PicoGreen fluorescence on a BioTek Synergy HTX microplate reader; only samples with > 5 ng/ul were selected. After 10ng of DNA from each high-quality extraction was plated, the 96-well plate was sealed with a microporous sealing film and stored at room temperature until liquid evaporated from all wells. DNA was then fragmented and tagged with a universal Nextera overhang following the Nextera DNA Library Prep Kit (Illumina, Inc., San Diego, CA) with some modifications (i.e., using 1/20th of recommended reagents). Tagmented libraries were then amplified with low-cycle PCR and barcoded using Illumina Nextera dual-indices at concentrations of 5uM. Additional amplification and the attachment of Illumina P5 and P7 sequencing primers was carried out using another round of low-cycle PCR. Tagmented and indexed samples were then normalized (<=25ng) using 96-well SequelPrep Normalization Plates following manufacturer protocol and then pooled for each plate. Pooled libraries were cleaned using AMPure XP beads (Beckman Coulter, Inc., Brea, CA) and eluted in 20ul of TLE buffer. Final lcWGS sequencing libraries were then visualized on an E-Gel to determine whether the ideal size range (200-1000bp) was achieved and quantified using a Qubit 2.0 dsDNA HS Assay (ThermoFisher, Inc., Waltham, MA). Four lcWGS libraries, each containing 96 individuals, were sequenced on two lanes of 2 x 150bp paired-end Illumina NovaSeq 6000 at the University of Oregon Genomics and Cell Characterization Core Facility.