Next-generation sequencing
Next-generation sequencing (NGS) was conducted using Illumina MiSeq for
every round of DNA library (0 to 4 rounds). Prior to sequence library
preparation, quantification was carried out using Qubit 2.0 Fluorometer
and Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific). A total 50
ng of the DNA library sample was used as an input for the NEBNext Ultra
II DNA Library Prep with Sample Purification Beads (NEB). We also used
NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) (NEB) to
multiplex our samples. The length of prepped DNA Library was determined
by using Agilent 2100 Bioanalyzer (Agilent Technologies), quantified
using Qubit 2.0 Fluorometer and run on MiSeq illumina platform using the
Miseq Reagent Kit v3 600 cycles.