Whole genome-seq of M.smegmatis
Next generation sequencing library preparations were constructed following the manufacturers protocol. For each sample, 100 ng genomic DNA was randomly fragmented to <500 bp by sonication (Covaris S220). The fragments were treated with End Prep Enzyme Mix for end repairing, 5 Phosphorylation and dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using VAHTSTM DNA Clean Beads, and fragments of ~470 bp (with the approximate insert size of 350 bp) were recovered. Each sample was then amplified by PCR for using P5 and P7 primers, with both primers carrying sequences which can anneal with flowcell to perform bridge PCR and P5/P7 primer carrying index allowing for multiplexing. The PCR products were cleaned up using DNA Clean Beads, validated using an Qsep100 (BiopticTaiwanChina), and quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).Then libraries with different indices were multiplexed and loaded on an Illumina HiSeq/Novaseq instrument according to manufacturers instructions (Illumina, San Diego, CA, USA) or a MGI2000 instrument according to manufacturers instructions (MGI, Shenzhen, China) . Sequencing was carried out using a 2x150 paired-end (PE) configuration; image analysis and base calling were conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrumentimage analysis and base calling were conducted by the NovaSeq Control Software (NCS) + OLB + GAPipeline-1.6 (Illumina) on the NovaSeq instrumentimage analysis and base calling were conducted by the Zebeacall on the MGI2000 instrument.