Hiseq x ten reagent kit v2

A single colony of each CIP-CAZ-CT co-resistant isolate was cultured overnight in brain heart infusion broth (Beijing Landbridge, China) at 37°C. Genomic DNA was extracted and purified using a TIANamp Bacterial DNA extraction kit (DP302, TIANGEN BIOTECH, China) and then sequenced with the SMRT^® Pacific Biosciences (PacBio) RS II platform (Tianjin Biochip Corporation, Tianjin, China) with a 10-kbp template library preparation step with PacBio^® Template Prep Kit. SMRT Analysis v2.3.0 was used for de novo assembly according to RS Hierarchical Genome Assembly Process (HGAP) workflow v3.0. Subsequently, Consed version 28.0 was used to manually inspect and trim duplicate ends to generate single, complete, and closed sequences for each chromosome and plasmid. For data error correction, Pilon v1.23 was used with Illumina MiSeq sequencing read data, for which a library was prepared with a NEBNext^® Ultra DNA Library Prep Kit for Illumina (NEB#E7370) followed by sonication fragmentation (350 bp insert), and then loaded on the Illumina HiSeq platform with PE 150 sequencing strategy (Novogene, Beijing, China) using a HiSeq X Ten Reagent Kit v2.5 (Illumina, San Diego, CA). The closed genomes were deposited in National Center for Biotechnology Information (NCBI) and automatically annotated using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAP, version 4.8).

Genomic DNA (1 μg) was randomly sheared to 350-bp fragments with Covaris cracker (Covaris) followed by sequencing library preparation using the Truseq Nano DNA HT Library Prep kit (Illumina). Sequencing libraries were sequenced paired-end 150 bp on the HiSeq X Ten sequencing platform (Illumina) with the HiSeq X Ten Reagent Kit v2.5 (Illumina) to a mean depth of coverage of about 50x. Reads were mapped to GRCh38 genome assembly using BWA-0.7.17 [53 (link)] with the default BWA-MEM parameters and 24 threads. SAM files were processed to sorted and indexed BAM files using SAMtools [52 (link)]. 2GS simulated reads were mapped to GRCh37 genome assembly using Minimap2 with the “-ax sr” parameter. For SV calling with novoBreak [26 (link)] (version 1.1.3rc), sorted and indexed BAM files were input with default run parameters. A dummy BAM file was simulated (GRCh38) to be used as a matched normal control. The confidence score for each breakend was obtained from the QUAL scores in the output VCF file. For SV calling with Delly [27 (link)] (version 0.7.8), duplicated reads in the BAM files were identified by Picard MarkDuplicates [54 ] before running Delly with the provided hg38 exclude file and its default parameters.

Whole-genome sequencing was performed at the University of Washington Center for Mendelian Genomics (University of Washington, Seattle). Initial quality control (QC) entailed DNA quantification, gender validation assay, and molecular fingerprinting with a 63-SNP OpenArray assay derived from a custom exome SNP set. Following successful QC, at least 750 ng of genomic DNA was subjected to a series library construction steps utilizing the KAPA Hyper Prep kit (Roche), automated on the Perkin Elmer Janus platform. Libraries were validated using the Bio-Rad CFX384 Real-Time System and KAPA Library Quantification kit (Roche). Samples were sequenced on a HiSeq X using Illumina’s HiSeq X Ten Reagent Kit (v2.5) to an average depth of 30X. Burrows-Wheeler Aligner^{52 (link)}, Genome Analysis ToolKit^{53 (link)} and SeattleSeq Annotation server build 138 (https://snp.gs.washington.edu/SeattleSeqAnnotation138/) were used to generate BAM, vcf and annotation files, respectively. Homozygosity mapping was performed with PLINK v1.07 software^{54 (link)} using the genotypes generated by the 63-SNP OpenArray assay. Structural variants were called using Lumpy^{55 (link)}. Alignments were visualized using the Integrative Genomics Viewer tool^{56 (link)}. The LMBR1 deletion and ZRS variants were validated by PCR-Sanger sequencing (primers provided in Supplementary Table 3).