Open AccessOptimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples
Author(s) -
Teresa Street,
Leanne Barker,
N Sanderson,
James Kavanagh,
Sarah Hoosdally,
Kevin Cole,
Robert Newnham,
Mathyruban Selvaratnam,
Monique Andersson,
Martin J. Llewelyn,
Justin O’Grady,
Derrick Crook,
David W Eyre
Publication year - 2020
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.01822-19
Subject(s) - neisseria gonorrhoeae , gonorrhea , dna extraction , nanopore sequencing , dna sequencing , neisseriaceae , biology , antibiotic resistance , microbiology and biotechnology , dna , transmission (telecommunications) , urine , nanopore , neisseria , polymerase chain reaction , antibiotics , computational biology , virology , bacteria , genetics , gene , computer science , nanotechnology , materials science , telecommunications , biochemistry , human immunodeficiency virus (hiv)
Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae -spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥10 4 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.