Smrt link software version 9

All isolates that arrived at the laboratory before or on 08/21/2020 were sequenced on the Illumina sequencing platform on a single MiSeq instrument (Illumina, San Diego, CA, USA). WGS library preparation, sequencing and subsequent data analysis were performed as described in [23 (link)]. Briefly, resulting fastq files were de novo assembled using SKESA. Isolates that arrived after 08/21/2020 were sequenced using the PacBio Sequel II system (Pacific Biosciences Inc., Menlo Park, CA, USA). WGS library preparation, sequencing and subsequent data analysis were performed as described recently [24 (link)] with minor modifications. After DNA extraction using the NEB Monarch Genomic Purification Kit (New England Biolabs, Ipswich, Massachusetts, USA), we constructed the sequence library using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences Inc.) in accordance with the manufacturer’s recommendations. After the 15 h-sequencing run on the Sequel II system, the resulting long reads were assembled by applying the “Microbial Assembly” pipeline within the SMRT Link software version 9 (Pacific Biosciences Inc.) using default parameters except for the genome size, which was adopted to 5.4 Mb to reflect the anticipated genome size of Bcg. More details regarding sequencing results are provided in Additional file 6: Table S6.

Bacterial genomic DNA was subjected to WGS on either a MiSeq platform (Illumina Inc., San Diego, CA, USA) or a Sequel II platform (Pacific Biosciences Inc., Menlo Park, CA, USA) at the Institute of Hygiene, Münster, Germany. WGS on the MiSeq platform and subsequent de novo assembly were performed as recently described (51 (link)). WGS on the Sequel II platform was conducted as follows: starting with approximately 1 μg of bacterial DNA, we constructed the sequence library using the SMRTbell Express template prep kit 2.0 (Pacific Biosciences Inc.) according to the manufacturer’s recommendations and subsequently loaded the library onto the Sequel II system for a 15-h data collection run. The resulting long-read sequencing data were then assembled, applying the microbial assembly pipeline within the SMRT Link software version 9 (Pacific Biosciences Inc.), using default parameters except for the genome size, which was adopted to the anticipated genome size of the sequenced species (e.g., 3.8 Mb for A. baumannii).