Optimizing DNA extraction methods for Nanopore sequencing of Neisseria gonorrhoeae direct from urine samples

Abstract

20 2 Oxfordshire Sexual Health Service, Oxford University Hospitals NHS Foundation Trust, 21 Oxford, UK g 22 Brighton and Hove sexual health and contraception service, Royal Sussex County Hospital, 23 Brighton, UK h 24 25 26 Running title: Nanopore sequencing N. gonorrhoeae direct from urine 27 Address correspondence to teresa.street@ndm.ox.ac.uk and david.eyre@bdi.ox.ac.uk. 28 3 Abstract 29Background. Empirical gonorrhoea treatment at initial diagnosis reduces onward 30 transmission. However, increasing resistance to multiple antibiotics may necessitate waiting 31 for culture-based diagnostics to select an effective treatment. There is a need for same-day 32 culture-free diagnostics that identify infection and detect antimicrobial resistance. 33 Methods. We investigated if Nanopore sequencing can detect sufficient N. gonorrhoeae 34 DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae 35 spiked urine samples and samples from gonorrhoea infections to determine optimal DNA 36 extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced whilst 37 minimizing contaminating host DNA. 38 Results. In simulated infections the Qiagen UCP Pathogen Mini kit provided the highest ratio 39 N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA 40 with saponin increased N. gonorrhoeae yields in simulated infections, but decreased yields 41 in clinical samples. In ten urine samples from men with symptomatic urethral gonorrhoea, 42 ≥87% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with 43 ≥92% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections if ≥10 4 44 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was 45 frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR Media 46 tubes and from urethral swabs, and in the presence of simulated Chlamydia co-infection. 47 Conclusion. Using Nanopore sequencing of urine samples from men with urethral 48 gonorrhoea sufficient data can be obtained to reconstruct whole genomes in the majority of 49 samples without the need for culture. 50 51 4 52

Application of respiratory metagenomics for COVID-19 patients on the intensive care unit to inform appropriate initial antimicrobial treatment and rapid detection of nosocomial transmission

“…Clinical metagenomic (CMg) using nanopore technology has potential to meet these needs due to its unbiased pan-microbial coverage (18,19) and ability to provide real-time data acquisition and analysis (20). It has been evaluated for respiratory, urinary tract and prosthetic joint infections (20)(21)(22)(23), however, the full clinical potential required for laboratories and clinical teams to change their long-standing practice has not been demonstrated. We therefore prospectively assessed whether CMg testing of respiratory samples from COVID- 19 patients with suspected secondary bacterial or fungal pneumonia, could significantly improve their initial antimicrobial treatment and detect outbreaks affecting a large COVID-19 patient cohort across 7 ICUs.…”

High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic Nanopore sequencing

“…10,12,13 In addition, we also tested our final algorithm on 10 nanopore metagenomic sequences from N. gonorrhoeae positive urine samples obtained from men with symptomatic urethral gonorrhoea, described previously. 2 Cultured isolates from the same infections were sequenced with an Illumina MiniSeq, following the manufacturer's instructions, to allow for comparisons.…”

“…1 We have recently described that rapid long-read sequencing using the Oxford Nanopore Technologies (ONT, Oxford, UK) R9.4.1 platform offers the potential to detect and sequence near-complete N. gonorrhoeae genomes directly from urine samples. 2 This clinical metagenomic approach has the advantage that it does not require prior bacterial culture, which typically adds 2-3 days to diagnostic workflows and may not be available in all cases, particularly in settings where diagnostics are based on molecular testing alone. With analysis possible during sequencing, 3 it could potentially offer a same day diagnostic tool for gonorrhoea infection that can guide antimicrobial treatment.…”