Real-time analysis of nanopore-based metagenomic sequencing from infected orthopaedic devices.
BMC Genomics
; 19(1): 714, 2018 Sep 27.
Article
em En
| MEDLINE
| ID: mdl-30261842
ABSTRACT
BACKGROUND:
Prosthetic joint infections are clinically difficult to diagnose and treat. Previously, we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current gold standard microbiological diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we report a real-time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections but potentially useful for any microbial sequencing, and compare detection by direct-from-clinical-sample metagenomic nanopore sequencing with Illumina sequencing and standard microbiological diagnostic techniques.RESULTS:
DNA was extracted from the sonication fluids of seven explanted orthopaedic devices, and additionally from two culture negative controls, and was sequenced on the Oxford Nanopore Technologies MinION platform. A specific analysis pipeline was assembled to overcome the challenges of identifying the true infecting pathogen, given high levels of host contamination and unavoidable background lab and kit contamination. The majority of DNA classified (> 90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological diagnosis and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within minutes from initiation of sequencing.CONCLUSIONS:
We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data and use of this pipeline to show initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. The high proportion of human DNA in prosthetic joint infection extracts prevents full genome analysis from complete coverage, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling. The nine samples sequenced in this pilot study have shown a proof of concept for sequencing and analysis that will enable us to investigate further sequencing to improve specificity and sensitivity.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bactérias
/
Análise de Sequência de DNA
/
Metagenômica
/
Prótese Articular
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article