Hash-Based Core Genome Multilocus Sequence Typing for Clostridium difficile

J Clin Microbiol. 2019 Dec 23;58(1):e01037-19. doi: 10.1128/JCM.01037-19. Print 2019 Dec 23.

Abstract

Pathogen whole-genome sequencing has huge potential as a tool to better understand infection transmission. However, rapidly identifying closely related genomes among a background of thousands of other genomes is challenging. Here, we describe a refinement to core genome multilocus sequence typing (cgMLST) in which alleles at each gene are reproducibly converted to a unique hash, or short string of letters (hash-cgMLST). This avoids the resource-intensive need for a single centralized database of sequentially numbered alleles. We test the reproducibility and discriminatory power of cgMLST/hash-cgMLST compared to those of mapping-based approaches in Clostridium difficile, using repeated sequencing of the same isolates (replicates) and data from consecutive infection isolates from six English hospitals. Hash-cgMLST provided the same results as standard cgMLST, with minimal performance penalty. Comparing 272 replicate sequence pairs using reference-based mapping, there were 0, 1, or 2 single-nucleotide polymorphisms (SNPs) between 262 (96%), 5 (2%), and 1 (<1%) of the pairs, respectively. Using hash-cgMLST, 218 (80%) of replicate pairs assembled with SPAdes had zero gene differences, and 31 (11%), 5 (2%), and 18 (7%) pairs had 1, 2, and >2 differences, respectively. False gene differences were clustered in specific genes and associated with fragmented assemblies, but were reduced using the SKESA assembler. Considering 412 pairs of infections with ≤2 SNPS, i.e., consistent with recent transmission, 376 (91%) had ≤2 gene differences and 16 (4%) had ≥4. Comparing a genome to 100,000 others took <1 min using hash-cgMLST. Hash-cgMLST is an effective surveillance tool for rapidly identifying clusters of related genomes. However, cgMLST/hash-cgMLST generate more false variants than mapping-based approaches. Follow-up mapping-based analyses are likely required to precisely define close genetic relationships.

Keywords: Clostridium difficile; core genome MLST; quality assurance; whole-genome sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Clostridioides difficile / classification*
  • Clostridioides difficile / genetics*
  • Clostridium Infections / microbiology*
  • Computational Biology / methods
  • Genome, Bacterial*
  • Humans
  • Multilocus Sequence Typing*
  • Polymorphism, Single Nucleotide
  • Sequence Analysis, DNA
  • Whole Genome Sequencing