Targeted DNA enrichment and whole genome sequencing of Neisseria meningitidis directly from clinical specimens

Abstract

In England and Wales, approximately one half of all laboratory-confirmed meningococcal disease cases fail to yield a viable invasive isolate, primarily due to the use of antibiotics. Characterisation of non-culture meningococci has been restricted to the detection or sequencing of specific gene targets within clinical specimens. In this study we investigated the ability of the Agilent SureSelectXT kit to facilitate DNA enrichment and genome sequencing of meningococcal DNA within a small panel of blood and CSF specimens. A target-specific RNA oligonucleotide bait library was used to capture and enrich the bacterial DNA prior to next generation sequencing. A positive correlation between meningococcal DNA amount and genome coverage was observed with eight of the ten specimens producing genomes of acceptable quality. All commonly-used typing information derived from each acceptable non-culture genome matched those of an isolate from the same patient and the paired genomes showed a high level of congruence across indexed loci. We estimate that this technique could be used to perform whole genome sequencing on up to ∼45% of the positive specimens received by the Public Health England's Meningococcal Reference Unit. Further optimisation of the extraction and/or enrichment processes may, however, increase the proportion of non-culture cases from which quality genomes can be obtained.

Pathogen Genomics in Public Health

“…84 One solution may be to bypass culture, sequencing pathogen genomes directly from specimens. 38,85 Although already feasible for selected organisms in particular specimens, the methods are not yet practical for routine use.…”

Full Molecular Typing of Neisseria meningitidis Directly from Clinical Specimens for Outbreak Investigation

“…Thus, several commercial kits have been developed to enrich bacterial DNA in clinical specimens by removing human DNA based on the differences between bacterial and human cell envelopes (15) or between bacterial and human DNA (16). An alternative enrichment strategy uses sequence-specific oligonucleotides to selectively pull-down bacterial DNA, therefore increasing the bacterial/human DNA ratio, as has been demonstrated with Nm RNA bait libraries (17). DNA enrichment can also be achieved using an isothermal multiple displacement amplification reaction with phi29 polymerase and primers designed to selectively amplify a microbial genome.…”

“…The reference tree for EPA ( Fig. 4) (15)(16)(17). SWGA has been successfully developed and used for pathogen genome sequencing in a number of applications for various types of specimens (18)(19)(20)(21)(22).…”

Hybrid Capture-Based Next Generation Sequencing and Its Application to Human Infectious Diseases

“…Other studies have used biotinylated baits spanning entire genomes for high-resolution strain genotyping directly from clinical samples. Indeed, discrimination of Chlamydia trachomatis serovars from genital samples would facilitate the study of population structures and modes of transmission ( Christiansen et al, 2014 ) while genomic data from uncultured Neisseria meningitidis not grown in the case of invasive meningococcal would allow increased surveillance of vaccine antigens and studies on possible vaccine deficiencies ( Clark S.A. et al, 2018 ).…”