Computational protocol: Complete Genome Sequence of Mycobacteriumchimaera Strain CDC2015-22-71

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Protocol publication

[…] Mycobacterium chimaera is a nontuberculous mycobacterium (NTM) species within the Mycobacterium avium complex (MAC). M. chimaera is an emerging cause of infection, in particular due to increasing awareness of infections linked with exposure to contaminated LivaNova Sorin Stockert 3T heater-cooler unit (HCU) devices (). Despite the public health relevance of M. chimaera, no publicly available genome is representative of genotypes implicated in HCU contamination (, ). We present this here, using CDC2015-22-71, a clinical isolate from a Pennsylvania patient epidemiologically linked to a cluster of M. chimaera infections following exposure to an HCU.Genomic DNA was extracted from CDC2015-22-71 grown in 7H9 broth (BD, Franklin Lakes, NJ, USA) at 36°C for approximately 1 week. The genome was sequenced using the Pacific Biosciences RSII (PacBio, Menlo Park, CA, USA) and Illumina MiSeq (San Diego, CA, USA) platforms. A 10-kb library was generated with the SMRTbell template prep kit 1. The library was then bound to polymerase using the DNA/polymerase binding kit P6v2. The bound library was loaded on two SMRTcells and sequenced with C4v2 chemistry for 360-min movies on the RSII instrument. Sequence reads were filtered and assembled de novo utilizing the PacBio HGAP version 3 (). The PacBio assembly was corrected with Illumina reads using Pilon version 1.20 (). The resulting assembly was compared to other M. chimaera genomes using Mauve version 2.3.1 (). Genomic features were identified and annotated using the NCBI Prokaryotic Genome Annotation Pipeline.The genome of M. chimaera CDC2015-22-71 consists of four scaffolds, equaling 6,247,640 bp (6,078,351-bp chromosome; 97,267-bp plasmid; 39,887-bp plasmid; 32,135-bp plasmid) and a G+C content of 67.6%. A total of 5,795 coding sequences were predicted, including 5,627 protein-coding genes and 168 pseudogenes. Our genome assembly contains 47 tRNAs, 3 noncoding RNAs, and 1 rRNA cistron consisting of the 16S, 23S, and 5S rRNA genes.Whole-genome alignments of M. chimaera CDC2015-22-71, Hawaiian respiratory strain AH16 (GenBank accession number CP012885 []), and Irish respiratory strains MCIMRL6, MCIMRL4, and MCIMRL2 (GenBank accession numbers LJHN00000000, LJHM00000000, and LJHL00000000 []) revealed 15,077 single-nucleotide polymorphisms (SNPs) compared to AH16; 8,870 SNPs compared to MCIMRL2; 2,229 SNPs compared to MCIMRL4; and 8,098 SNPs compared to MCIMRL6. Comparison of gene content between the five M. chimaera genomes revealed a core gene set of 4,726 genes (83.9% of the 2015-22-71 genome []). The comparison of CDC2015-22-71 against all previously identified M. chimaera genomes resulted in average nucleotide identity values greater than or equal to 98.70%, which are greater than the 95 to 96% cutoff for species boundaries ().This M. chimaera genome assembly is the first complete NTM genome associated with an outbreak and will serve as a reference for epidemiological investigations of North American-based HCU contamination and postsurgical M. chimaera infections. […]

Pipeline specifications

Software tools HGAP, Pilon, Mauve, PGAP
Application Nucleotide sequence alignment