Next Generation Sequencing (NGS)

Genomic DNA was fragmented to an average size of 250 bp in 40 µL volume of EB buffer (Qiagen) using Covaris E-220 Focused-ultrasonicator (55 sec, 20% duty factor, 200 bursts/min). All liquid handling steps were carried out on the Bravo liquid handling platform using in house created programs with VWorks Automation Control Software (Agilent Automation, Santa Clara, CA, USA). End Repair and 5′ Phosphorylation reaction (40 µL DNA sample, 5.5 µL of 10X NEB 2 buffer, 2.2 µL of 25 mM ATP, 2.2 µL of 10 mM dNTP, 10U T4 Polynucleotide Kinase, 4.5U T4 DNA Polymerase, 1U Klenow Large Fragment DNA Polymerase, and ultrapure water to a total reaction volume of 55 µL (New England Biolabs Ipswich, MA, USA) was incubated for 30 minutes at room temperature. DNA samples were then purified using in house prepared Sera-Mag magnetic bead solution (1 M NaCl, 23% PEG, Sera-Mag Speedbeads (Fisher Scientific, Pittsburgh PA, USA)) with final PEG concentration of 13.8% and eluted in 35 µL volume with Qiagen EB buffer. To enable ligation to the adaptors, a single dA overhang was added to the 3′ ends of DNA fragments (35 µL DNA, 5 µL of 10X NEB 2 buffer, 1 µL of 10 mM dATP, 5U Klenow Fragment (3′ → 5′ exo–), and ultrapure water to a total reaction volume of 50 µL, (New England Biolabs Ipswich, MA, USA)). The reaction was incubated at 37 °C for 30 minutes. The product was then purified using in house-made Sera-Mag magnetic bead solution with final PEG concentration of 13.8% and eluted in 35 µL volume with Qiagen EB buffer. Next, short adaptors containing sequences required downstream in the sequencing workflow were ligated to the dA-tailed DNA fragments (35 µL DNA, 12 µL of 5X Quick Ligation Buffer, 2000U Quick T4 DNA Ligase, 2 µL of 0.5 µM Illumina short sequencing adaptor, and ultrapure water to a total volume of 60 µL). Ligation reaction was performed at room temperature overnight. The ligation product was then purified using in house-made Sera-Mag magnetic bead solution with final PEG concentrations of 10.2% and eluted from the beads with 35 µL of Qiagen EB buffer.

Adaptor ligated libraries were PCR amplified and barcoded by custom indexing primers in a 60 µl PCR reaction (35 µL DNA, 12 µL of 5X High fidelity buffer, 1 µL of 10 mM dNTPs, 1.5 µL of DMSO, 1 µL of 25 µM PCR primer 1.0, 2 µL of 12.5 µM custom indexing primer (added separately to each well), 1U Phusion Hot Start II, and ultrapure water to a total volume of 60 µL (Fisher Scientific, Pittsburgh PA)). PCR amplification was carried out with the following cycling conditions: 98°C for 30 seconds, 6 cycles of (15 seconds at 98 °C (denaturation), 30 seconds at 65 °C (annealing), 30 seconds 72 °C (extension)), followed by 5 min at 72 °C. PCR amplified libraries were cleaned up with in house-made Sera Mag magnetic bead solution with final PEG concentration of 10.5% and eluted with 35 µL of Qiagen EB buffer. Libraries were quantified using Qubit HS DNA assay, pooled equal-molar for MiSeq sequencing. 3.7GB of data was generated in a PE 75 bp run with base quality of 97% of Q30 or above. Adapter trimming and alignment to sacCer3 genome was performed on the MiSeq with alignment rates ranging between 95–99%. Samtools V0.1.19 (https://www.ncbi.nlm.nih.gov/pubmed/19505943) was used to extract the number of reads per chromosome per sample. These counts were then normalized to the library depth by dividing by the total number of aligned reads per sample.