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 theSupplementary Materials (Text S1 ).
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