title: TD3
design: llumina-compatible sequencing library preparation for Bacterial sample was done at Genotypic Technology Pvt. Ltd. Genomics facility with NEXTFlex DNA Sequencing Kit (Bioo Scientific, # 5140-02) according to manufacturer recommended protocol. In brief, gDNA was sheared to generate fragments of approximate size 200bp - 400bp using Covaris S220 system (Life Technologies, #4465653). The fragment size distribution was confirmed on Agilent TapeStation with D1000 DNA Kit (Agilent, #5067-5583 (reagents) #5067-5582 (tapes)). Next, the fragmented DNA was purified using HighPrep beads (Magbio Genomics, #AC-60050) followed by end-repair, A-tailing, and ligation of the Illumina multiplexing adaptors (Bioo Scientific, #514104) according to protocol outlined in NEXTFlex DNA Sequencing kit. The adapter-ligated DNA was purified using HighPrep beads and then subjected to 10 rounds of PCR (denaturation at 98˚C for 2 min, cycling (98˚C for 30sec, 65˚C for 30sec and 72˚C for 60s) and final extension at 72˚C for 4 min) with Illumina-compatible primers provided with the NEXTFlex DNA Sequencing kit. Finally, the PCR product (Illumina-compatible sequencing library) was purified using HighPrep beads, followed by quantification with Qubit fluorometer (Table1) and fragment size distribution analysis on Agilent D1000 TapeStation (Figure 2). The Illumina-compatible sequencing library was quantified using KAPA qPCR quantification kit (KAPA Biosystems; #KK4824) prior to loading on corresponding Illumina Sequencer flowcell.