Target enrichment improves culture-independent detection ofNeisseria gonorrhoeaedirect from sample with Nanopore sequencing
Authors: Street Teresa L. , Sanderson Nicholas D. , Barker Leanne , Kavanagh James , Cole Kevin ,   The GonFast Investigators Group, Llewelyn Martin , Eyre David W. 
Published: January 2024

AbstractMulti-drug resistantNeisseria gonorrhoeaeinfection is a significant public health risk. Rapidly detectingN. gonorrhoeaeand antimicrobial resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high levels of host DNA and overgrowth of contaminating species hamper sequencing and limitN. gonorrhoeaegenome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel to investigate selectively enriching forN. gonorrhoeaeDNA. Probes were designed to cover the entireN. gonorrhoeaegenome, with 10-fold enrichment of probes covering selected AMR determinants. Multiplexing was tested in subset of samples. The proportion of sequence bases classified asN. gonorrhoeaeincreased in all samples after enrichment, from a median (IQR) of 0.05% (0.01-0.1%) to 76% (42-82%), giving a corresponding median improvement in fold genome coverage of 365-times (112–720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15 (87%) samples, compared to 2/15 (13%) without enrichment. The four samples multiplexed together also achieved >20x genome coverage. Coverage of AMR determinants was sufficient to predict resistance where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improveN. gonorrhoeaegenome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for robust detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.Impact statementNeisseria gonorrhoeaeinfection presents a significant public health risk, with multi-drug resistance present globally. Early detection helps control the spread of antimicrobial resistant strains. Genome sequencing can be used to detect infections in samples collected directly from patients, without the need to grow any microorganisms in a laboratory first, and this has already been demonstrated for gonorrhoea using urine samples. With enough sequence information it is also possible to detect antimicrobial resistance (AMR), allowing both detection of infection and information on treatment choices from the same test. This study assesses a method for enrichingN. gonorrhoeaesequence data from urine and urethral swabs, and analyses the impact of enrichment on the detection of genes known to cause antibiotic resistance. We show that enriching forN. gonorrhoeaeDNA prior to sequencing can improve the detection of some AMR genes, and by testing several samples at the same time we can reduce the costs associated with this method.Data summaryThe sequence data generated in this study are deposited in the European Nucleotide Archive (ENA,https://www.ebi.ac.uk/ena/browser/) and are publicly available under study code PRJEB64347. The authors confirm all supporting data, code and protocols are provided within the article or through supplementary data files.