DNA-Seq of Enterobacter cloacae cpx: Clinical isolate
Genomic DNA was isolated from 96 Enterobacter clinical isolates and 8 NR3055 SR clones using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI), following the manufacturers protocol for isolating genomic DNA from Gram-negative bacteria. Bacterial whole genome library preparation was performed using the Nextera DNA Flex Library Prep Kit (96rxn kit, cat# 20025520, Illumina, San Diego, CA). DNA concentrations were verified using the Quant-iT High Sensitivity dsDNA Assay Kit (cat# Q33120, ThermoFisher, Waltham, MA) and a microplate reader. The library preparation is a bead-based tagmentation reaction which cuts the dsDNA (100 ng input each normalized to 30 L volume) to roughly 350 base pair (bp) size and all subsequent library preparation steps are done on the beads. The protocol was modified to include the use of one quarter scale reaction mixes for all reactions throughout the entire library preparation procedure. Following PCR amplification (five cycles total), according to the protocol, water was added (38 L) to the amplified material (12.5 L) to raise the volume up to 50 L for the final 1x Ampure XP bead cleanup (cat# A63882, Beckman Coulter Life Sciences, Indianapolis, IN). Library DNA was run on the TapeStation 2200 with high sensitivity DNA screentape (Agilent Technologies, Santa Clara, CA) to verify a library size of approximately 500 bp to 650 bp and each individual library was run on qPCR for library quantitation using the Kapa-Roche Library quant kit (cat# KK4824, Roche) on the CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Sequencing was run on the NovaSeq 6000 System (Illumina, San Diego, CA) using a full SP300 cartridge and flow cell (paired end 150 dual indexing run), resulting in ~ 250x coverage.