Public health microbiology laboratories (PHLs) are on the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole-genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards. Here, we describe the validation of WGS on the Illumina platform for routine use in PHLs according to Clinical Laboratory Improvements Act (CLIA) guidelines for laboratory-developed tests (LDTs). We developed a validation panel comprising 10 Enterobacteriaceae isolates, 5 Gram-positive cocci, 5 Gram-negative nonfermenting species, 9 Mycobacterium tuberculosis isolates, and 5 miscellaneous bacteria. The genome coverage range was 15.71× to 216.4× (average, 79.72×; median, 71.55×); the limit of detection (LOD) for single nucleotide polymorphisms (SNPs) was 60×. The accuracy, reproducibility, and repeatability of base calling were >99.9%. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from multilocus sequence typing (MLST) and genome-wide SNP-based phylogenetic assays were 100%. The following objectives were accomplished: (i) the establishment of the performance specifications for WGS applications in PHLs according to CLIA guidelines, (ii) the development of quality assurance and quality control measures, (iii) the development of a reporting format for end users with or without WGS expertise, (iv) the availability of a validation set of microorganisms, and (v) the creation of a modular template for the validation of WGS processes in PHLs. The validation panel, sequencing analytics, and raw sequences could facilitate multilaboratory comparisons of WGS data. Additionally, the WGS performance specifications and modular template are adaptable for the validation of other platforms and reagent kits.

T uricella otitidis is a nonfermenting Gram-positive non-sporeforming rod usually isolated from ear exudates. The bacterium was described originally in 1994 from acute and chronic otitis media and has since been isolated from a posterior auricular abscess, mastoiditis, and blood (1-3). To date, only one case of bacteremia due to T. otitidis has ever been reported (3). Its association with otitis media is controversial, as it has been isolated from the external auditory canal of healthy subjects at ratios comparable to those noted when T. otitidis otitis media was diagnosed (4-6). Most isolates demonstrate high resistance to macrolides and lincosamides with associated 23S rRNA mutations (4, 7).As only one whole genome of T. otitidis had been sequenced previously, we sequenced the draft genome of T. otitidis strain TD1, isolated from a central line catheter tip culture. The isolate, and another one on the same patient, from the same source, that was submitted 3 months before, had previously been identified in our laboratory by biochemical and cellular fatty acid analysis.DNA from strain TD1 was extracted using the Promega Genomic DNA Wizard kit, and paired-end libraries were constructed using the Nextera XT kit. Sequences were adapted and quality (Q30) trimmed using Cutadapt, de novo assembled using SPAdes version 3.5, metagenomically screened with SURPI, and annotated via Prokka version 1.1, as described previously (8-12). A total of 739, 492 paired-end reads with an average length of 173 nucleotides were recovered after trimming. De novo assembly yielded 160 contigs covering a total of 2,150,112 bp with an N 50 of 24,176 bp, a GC content of 71.2%, an average coverage of 54ϫ, and a total of 1,866 annotated coding sequences.BLASTn of the assembled 16S sequence from strain TD1 against the NCBI WGS database aligned 99.35% to T. otiditis ATCC 51513 and had an average nucleotide identity of 98.75% to ATCC 51513, confirming the identity of the strain (13). Direct mapping of TD1 reads to ATCC 51513 revealed 20,176 variants between the two strains. De novo assembly of reads that failed to map to ATCC 51513 revealed a total of 85.4 kb unique sequence, including a locus of 50 kb containing Ͼ60 hypothetical proteincoding sequences with no nucleotide homology to the NT database that aligned Ͻ35% amino acid by BLASTx and HHPred to various mycobacterium phage protein sequences (14). Genes in the remaining loci not present in ATCC 51513 included an arylsulfatase, a cadmium-cobalt antiporter, a phosphate/phosphonate transporter operon, and a unique ABC transporter, with all predicted proteins having Ͻ65% amino acid identity to the closest species, Corynebacterium glycinophilum AJ 3170. Antibiotic resistance genes annotated by the Comprehensive Antibiotic Resistance Database included a cfrA 50S methyltransferase (99% amino acid to T. otitidis ATCC 51513) and two major facilitator superfamily-type drug-resistance transporters (99% amino acid T. otitidis ATCC 51513 and 57 to 59% amino acid to Corynebacterium spp.) (15). In addition...

We report the first draft genome sequence of Kerstersia gyiorum from a leg ulcer of a patient with diabetes and osteomyelitis. The 3.94-Mb genome assembly included 3,428 annotated coding sequences with an N50 of 223,310 bp and a plasmid encoding a type IV secretion system gene and two antitoxin genes.

BackgroundPublic health microbiology laboratories (PHL) are at the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards.Objectives1) Establish the performance specifications of WGS applications used in PHL to conform with CLIA (Clinical Laboratory Improvements Act) guidelines for laboratory developed tests (LDT), 2) Develop quality assurance (QA) and quality control (QC) measures, 3) Establish reporting language for end users with or without WGS expertise, 4) Create a validation set of microorganisms to be used for future validations of WGS platforms and multi-laboratory comparisons and, 5) Create modular templates for the validation of different sequencing platforms.MethodsMiSeq Sequencer and Illumina chemistry (Illumina, Inc.) were used to generate genomes for 34 bacterial isolates with genome sizes from 1.8 to 4.7 Mb and wide range of GC content (32.1%-66.1%). A customized CLCbio Genomics Workbench - shell script bioinformatics pipeline was used for the data analysis.ResultsWe developed a validation panel comprising ten Enterobacteriaceae isolates, five gram-positive cocci, five gram-negative non-fermenting species, nine Mycobacterium tuberculosis, and five miscellaneous bacteria; the set represented typical workflow in the PHL. The accuracy of MiSeq platform for individual base calling was >99.9% with similar results shown for reproducibility/repeatability of genome-wide base calling. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from MLST and genotyping tests were 100%. A test report format was developed for the end users with and without WGS knowledge.ConclusionWGS was validated for routine use in PHL according to CLIA guidelines for LDTs. The validation panel, sequencing analytics, and raw sequences will be available for future multi-laboratory comparisons of WGS in PHL. Additionally, the WGS performance specifications and modular validation template are likely to be adaptable for the validation of other platforms and reagents kits.