title: WGS of Strix occidentalis caurina: adult female blood
design: We extracted genomic DNA from blood using a DNeasy Blood & Tissue Kit (Qiagen). We sheared 4,580 ng genomic DNA in 130 µL in a microTUBE AFA Fiber Pre-Slit Snap-Cap tube (Covaris) using a M220 focused-ultrasonicator (Covaris) targeting 900 bp as the center of the fragment distribution. We used peak incident power 50 W, 5% duty factor, 200 cycles per burst, and 70 s treatment time at 20°C. We then removed small fragments and concentrated the sheared material using a DNA Clean & Concentrator -5 kit (Zymo Research). We next constructed a genomic library by using a TruSeq DNA PCR-Free Library kit (Illumina) and following the manufacturer's protocol, except that we only performed a bead-based size selection to remove small fragments and not large fragments. We used a 0.45X bead to sample ratio in order to eliminate fragments smaller than approximately 700 bp. Following A-tailing and prior to adapter ligation, we took 10% of the sample (by volume) and separated it from the noPCR aliquot for use in a PCR-amplified library. We ligated adapters to these two aliquots separately and cleaned the finished ligations with a DNA Clean & Concentrator -5 kit (Zymo Research). We then only went forward with the aliquot for use in a PCR-amplified library. We used a BluePippin (Sage Science) to select library fragments in the size range of 800-1100 bp, which, after subtracting the 121 bp of adapters, corresponded to selecting an average insert size of 829 bp. We next cleaned the eluted material with a DNA Clean & Concentrator -5 kit (Zymo Research) and then performed real-time PCR (rtPCR) using a KAPA Real-Time Library Amplification Kit (KAPA Biosystems) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) to amplify the library. We amplified the library with 11 cycles PCR and then cleaned the PCR products with 1X Agencourt AMPure XP (Beckman Coulter) magnetic beads. We pooled the final library with two other libraries equimolarly and we obtained 350 bp read 1 and 250 bp read 2 for a total of 600 bp of paired-end read data from one lane (approximately 1/3 of one lane for this library) using a 600-cycle MiSeq Reagent Kit v3 on a MiSeq (Illumina).