Whole genome sequencing in clinical and public health microbiology

Abstract

Genomics and whole genome sequencing (WGS) have the capacity to greatly enhance knowledge and understanding of infectious diseases and clinical microbiology.The growth and availability of bench-top WGS analysers has facilitated the feasibility of genomics in clinical and public health microbiology.Given current resource and infrastructure limitations, WGS is most applicable to use in public health laboratories, reference laboratories, and hospital infection control-affiliated laboratories.As WGS represents the pinnacle for strain characterisation and epidemiological analyses, it is likely to replace traditional typing methods, resistance gene detection and other sequence-based investigations (e.g., 16S rDNA PCR) in the near future.Although genomic technologies are rapidly evolving, widespread implementation in clinical and public health microbiology laboratories is limited by the need for effective semi-automated pipelines, standardised quality control and data interpretation, bioinformatics expertise, and infrastructure.

Fig. 1

Whole genome sequencing workflow. (1) DNA extraction from homogeneous microbial samples, e.g., single bacterial colony from a pure culture. (2) Whole genome sequencing using next-generation sequencers. Most high-throughput sequencers produce short reads (e.g., Illumina MiSeq), although long reads from Pacific Biosciences RS II or Illumina TruSeq technology may facilitate de novo assembly more readily. (3) SNPs called from read mapping to a reference genome can be used for phylogenetic comparisons to assist in epidemiological and outbreak analyses. Reads can also be assembled de novo into longer contiguous sequences (contigs), and orientated and aligned to form scaffolds. (4) The resulting de novo assemblies can be used for further analyses such as typing and resistance detection based on local alignment tools (e.g., BLAST), or can be further finished into a completed or closed genome. This finishing stage usually requires gap closure through extensive ‘wet-lab’ techniques such as primer walking, and so is generally performed for research purposes, although WGS long reads are increasingly being used to produce more complete de novo assemblies and minimise the amount of laboratory work required. (5) Data analysis for outbreak investigation, typing, or resistance detection. Closed annotated genomes can be used as reference genomes for comparison, or can be analysed in further detail.

Fig. 2

Key considerations in quality assessment of whole genome sequencing analyses. Contigs, contiguous sequences; GC, genome coverage; SNP, single nucleotide polymorphism; wgMLST, whole genome multi-locus sequence typing.