Afa microtube

Each pool was sheared on a Covaris M220 (Covaris, Woburn, MA, USA) in a 130 μL Covaris Adaptive Focused Acoustics (AFA) microtube (Covaris, Woburn, MA, USA) to a target size of ~350–400 bp, with the following settings: 50 W peak incident power, 20% duty factor, 200 cycles per burst, 65 s treatment time.
Successful fragmentation was checked by running 1 μL of the sheared pool with a Bioanalyzer High-Sensitivity DNA kit on a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA, USA).
The validated pools were then processed with the TruSeq DNA Nano Low-Throughput Library Prep kit and TruSeq DNA Single Index Set A and B, according to the manufacturer’s protocol (Illumina, San Diego, CA, USA).
Molarities of the libraries were calculated and diluted to a 4 nM working concentration. The different libraries that were to be sequenced together (with different indexes) were pooled proportionally to their total genomic target size. Subsequently, these pools were denatured with 0.2N NaOH and loaded into a MiSeq Reagent Kit V2 (300 cycles) cartridge, according to the manufacturer’s recommendations (Illumina, San Diego, CA, USA). Paired-end sequencing (2× 151 cycles) was performed on a MiSeq instrument (Illumina, San Diego, CA, USA).
A detailed step-by-step protocol is provided in the Supplementary Materials (Text S1).

Three 3 μg aliquots of the same genomic DNA were each made up to a total volume of 130μl with 10 mM Tris-HCl, pH 8.0. After mixing, each was transferred to a separate Covaris AFA microTUBE with pre-split snap-cap (Product number 520045) and sheared to an average size of approximately 200 bp using a Covaris S2 focused-ultrasonicator (duty cycle 10%, intensity 5, 200 cycles per burst for 6 × 60s using frequency sweeping). The size distribution of the fragmented DNA was assessed with an Agilent 2100 Bioanalyser using a high sensitivity DNA chip. Each DNA aliquot was then purified using 1.4 × AxyPrep Mag PCR Clean-Up beads (Axygen) with two 70% ethanol washes (400 μl) and elution in 50 μl of nuclease-free water (Ambion). Each aliquot of purified fragmented DNA was used as input material for standard SureSelect library preparation or SureSelect Methyl-Seq library preparation as described below.