Enrichriching Neisseria gonorrhoeae genomes with sureselect probes and sequencing with Oxford nanopore

Background: Multi-drug resistant Neisseria gonorrhoeae infection is a significant public health risk. Transmission and spread of resistance are potentially increased by delays waiting for current culture-based diagnostics. Rapidly detecting N. gonorrhoeae and antimicrobial resistant (AMR) determinants by metagenomic sequencing of urine is possible. However, high levels of host DNA and overgrowth of contaminating species hamper sequencing and limit N. gonorrhoeae genome coverage. We performed Nanopore sequencing of urine and urethral swabs with and without probe-based target enrichment using a custom SureSelect panel to investigate selectively enriching for N. gonorrhoeae DNA. Materials/methods: SureSelect probes were designed to cover the entire N. gonorrhoeae genome, with 10-fold enrichment of probes covering selected AMR determinants. DNA, extracted from N. gonorrhoeae nucleic acid amplification test-positive urines and culture-positive urethral swabs, was hybridised with probes overnight. DNA was sequenced on an Oxford Nanopore GridION by Ligation Sequencing (SQK-LSK110) (with probe-based enrichment) or by Rapid PCR Barcoding (SQK-RPB004) (no enrichment). All samples were sequenced individually on R9.4.1 flow cells. Multiplexing was tested in subset of samples: DNA was barcoded using the Rapid PCR Barcoding kit (SQK-RPB004) and pooled prior to hybridisation. Sequence reads were analysed using the CRuMPIT pipeline. Results: N. gonorrhoeae-positive urine samples (n=11) and urethral swabs (n=4) were sequenced with and without target enrichment. Sequence bases classified as N. gonorrhoeae increased in all samples after enrichment, from a median (IQR) of 5.0Mb (1.7Mb-11.2Mb) to 6.6Gb (1.7Gb-14.5) (Fig.1), giving a corresponding improvement in fold genome coverage of 266-times (51-716). 13/15 (87%) achieved over the 20x coverage required for robust AMR determinant detection, compared to 2/15 (13%) without enrichment. 2/15 (13%) failed to reach 20x coverage after enrichment. Four samples were also sequenced multiplexed: 2/4 (50%) achieved >20x coverage and 2/4 (50%) achieved >10x coverage (11.6x and 16.9x). Conclusions: Probe-based target enrichment can improve N. gonorrhoeae genome coverage when sequencing DNA extracts direct from urine/urethral swabs, allowing for robust detection of AMR determinants. Multiplexing prior to enrichment could still provide enough data for AMR detection and would reduce the costs associated with this method. Results of AMR detection and relatedness/transmission from enriched samples will also be presented.