Illumina amplicon sequencing of Escherichia coli: VIM-2 plasmid library
We grouped individual codon libraries into seven groups of 39 consecutive codons (33 for the last group) so that all mutations are within a distance of 117 bp (99 bp for the last group), allowing 150 bp forward and reverse NGS sequencing reads to generate full overlap of each group. PCR amplicons of each library group and wtVIM-2 were generated using primers that flank the 117 bp region of each group, where primers have a Nextera transposase adapter sequence (Illumina, Inc.) in the 5 overhangs. We used KAPA HiFi PCR Kit (Kapa Biosystems, Inc.) for 15 cycles of amplification each with denaturation at 98C for 20 s, annealing at 65C for 5 s and no extension time. The amplicons were extended by PCR again to include the sample indices (i7 and i5) and flow cell binding sequence and then sequenced using a NextSeq 550 sequencing system (Illumina, Inc.).