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Whole genome sequencing

DS Devin Sindeldecker
MD Matthew Dunn
AZ Aubree Zimmer
MA Matthew Anderson
JA Juan Alfonzo
PS Paul Stoodley
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DNA extraction was performed for each of the isolated samples using the GenElute™ Bacterial Genomic DNA kit (Sigma-Aldrich). After gDNA extraction, samples were pooled to obtain samples of 400 ng gDNA to identify conserved driver mutations. Library preparation and barcoding was completed using the Rapid Barcoding Kit (Nanopore). A Nanopore MinION Sequencer was used to complete bacterial whole genome sequencing using default settings. After sequencing, quality control was performed using Epi2ME (Nanopore). Reads were trimmed using Porechop29 and aligned to a reference P. aeruginosa PAO1 genome (GCF_000006765.130) using graphmap31. Reads were sorted and indexed using samtools32. Structural variants were identified using sniffles33 and SNPs were identified using BCFtools34. SNPs were selected by a quality score > 20.

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