Comparing Gold-Standard Sanger Sequencing with Two Next-Generation Sequencing Platforms of HIV-1 gp160 Single Template Amplicons.

Our goal was to assess the accuracy of next generation sequencing (NGS) compared to Sanger. We performed single genome amplification (SGA) of HIV-1 gp160 on extracted tissue DNA from two HIV+ individuals. Amplicons (n=30) were sequenced with Sanger, or re-amplified with barcoded primers and pooled before sequencing using Oxford Nanopore Technologies [ONT] and Pacific Bioscience [PB]. For each amplicon, a consensus sequence for NGS reads was obtained by (1) mapping reads to the Sanger sequence when available ("reference-based") or (2) mapping reads to a "pseudo-reference" sequence, i.e., a consensus sequence of a subset of NGS reads ("reference-free"). PB reads were clustered based on genetic similarity. A Sanger consensus sequence was obtained for 23/30 amplicons, for which all NGS consensus sequences were identical [n=9] or nearly identical [n=14] compared to Sanger. For the nine mismatches between Sanger/NGS, the nucleotide in the NGS sequence matched all other sequences from that patient. Of the 7/30 amplicons without a Sanger sequence, NGS sequences had 35 ambiguous calls in five amplicons, and 0 ambiguities in two amplicons. Analysis of the electropherograms showed failure of a single sequencing primer for the latter two amplicons (consistent with a single template), and overlapping peaks for the other five (consistent with multiple templates). Clustering results closely followed the Sanger/NGS consensus results, where amplicons derived from a single template also had a single cluster, and vice versa (with one exception, which could be the result of barcode misidentification). Representative sequences from the clusters contained 2 -13 differences compared to Sanger/NGS. In summary, we show that both ONT and PB can produce amplicon consensus sequences with similar or higher accuracy compared to Sanger, and importantly, without the need for a known reference sequence. Clustering could be useful in some circumstances to predict or confirm the presence of multiple starting templates.

Whole-genome sequencing of carbapenem-resistant Enterobacterales isolates in southeast Louisiana reveals persistent genetic clusters spanning multiple locations.

BACKGROUND: We investigated 51 g-negative carbapenem-resistant Enterobacterales (CRE) isolates collected from 22 patients over a five-year period from six health care institutions in the Ochsner Health network in southeast Louisiana. METHODS: Short genomic reads were generated using Illumina sequencing and assembled for each isolate. Isolates were classified as Enterobacter spp. (n = 20), Klebsiella spp. (n = 30), and Escherichia coli (n = 1) and grouped into 19 different multi-locus sequence types (MLST). Species and patient-specific core genomes were constructed representing ∼50% of the chromosomal genome. RESULTS: We identified two sets of patients with genetically related infections; in both cases, the related isolates were collected > 6 months apart, and in one case, the isolates were collected in different locations. On the other hand, we identified four sets of patients with isolates of the same species collected within 21 days from the same location; however, none had genetically related infections. Genes associated with resistance to carbapenem drugs (blaKPC and/or blaCTX-M-15) were found in 76% of the isolates. We found three blaKPC variants (blaKPC-2, blaKPC-3, and blaKPC-4) associated with four different Enterobacter MLST variants, and two blaKPC variants (blaKPC-2, blaKPC-3) associated with seven different Klebsiella MLST variants. CONCLUSIONS: Molecular surveillance is increasingly becoming a powerful tool to understand bacterial spread in both community and clinical settings. This study provides evidence that genetically related infections in clinical settings do not necessarily reflect temporal associations, and vice versa. Our results also highlight the regional genomic and resistance diversity within related bacterial lineages.

Comparing antimicrobial resistant genes and phenotypes across multiple sequencing platforms and assays for Enterobacterales clinical isolates.

INTRODUCTION: Whole genome sequencing (WGS) of bacterial isolates can be used to identify antimicrobial resistance (AMR) genes. Previous studies have shown that genotype-based AMR has variable accuracy for predicting carbapenem resistance in carbapenem-resistant Enterobacterales (CRE); however, the majority of these studies used short-read platforms (e.g. Illumina) to generate sequence data. In this study, our objective was to determine whether Oxford Nanopore Technologies (ONT) long-read WGS would improve detection of carbapenem AMR genes with respect to short-read only WGS for nine clinical CRE samples. We measured the minimum inhibitory breakpoint (MIC) using two phenotype assays (MicroScan and ETEST) for six antibiotics, including two carbapenems (meropenem and ertapenem) and four non-carbapenems (gentamicin, ciprofloxacin, cefepime, and trimethoprim/sulfamethoxazole). We generated short-read data using the Illumina NextSeq and long-read data using the ONT MinION. Four assembly methods were compared: ONT-only assembly; ONT-only assembly plus short-read polish; ONT + short-read hybrid assembly plus short-read polish; short-read only assembly. RESULTS: Consistent with previous studies, our results suggest that the hybrid assembly produced the highest quality results as measured by gene completeness and contig circularization. However, ONT-only methods had minimal impact on the detection of AMR genes and plasmids compared to short-read methods, although, notably, differences in gene copy number differed between methods. All four assembly methods showed identical presence/absence of the blaKPC-2 carbapenemase gene for all samples. The two phenotype assays showed 100% concordant results for the non-carbapenems, but only 65% concordance for the two carbapenems. The presence/absence of AMR genes was 100% concordant with AMR phenotypes for all four non-carbapenem drugs, although only 22%-50% sensitivity for the carbapenems. CONCLUSIONS: Overall, these findings suggest that the lack of complete correspondence between CRE AMR genotype and phenotype for carbapenems, while concerning, is independent of sequencing platform/assembly method.