ena-STUDY-UNIVERSITY OF OXFORD-14-11-2017-16:11:33:443-183
ONT MinION sequencing of prosthetic joint infection sonication fluids
Prosthetic joint infections are clinically difficult to diagnose and treat. Previously we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we compare direct-from-clinical-sample metagenomic Illumina sequencing with Nanopore sequencing, and report a real time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections.DNA was extracted from the sonication fluids of nine explanted orthopaedic devices, including two culture negative controls, and were sequenced on the Oxford Nanopore MinION platform. With high levels of host contamination and unavoidable background lab and kit contamination, a specific analysis pipeline was assembled to overcome the challenges of identifying the true infection pathogen.The majority of DNA classified (>90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological services diagnoses and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within ten minutes from initiation of sequencing. We demonstrate initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data. The high proportion of human DNA in extracts prevents further genotyping, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling.
Prosthetic joint infections are clinically difficult to diagnose and treat. Previously we demonstrated metagenomic sequencing on an Illumina MiSeq replicates the findings of current diagnostic techniques. Nanopore sequencing offers advantages in speed of detection over MiSeq. Here, we compare direct-from-clinical-sample metagenomic Illumina sequencing with Nanopore sequencing, and report a real time analytical pathway for Nanopore sequence data, designed for detecting bacterial composition of prosthetic joint infections.DNA was extracted from the sonication fluids of nine explanted orthopaedic devices, including two culture negative controls, and were sequenced on the Oxford Nanopore MinION platform. With high levels of host contamination and unavoidable background lab and kit contamination, a specific analysis pipeline was assembled to overcome the challenges of identifying the true infection pathogen.The majority of DNA classified (>90%) was host contamination and discarded. Using negative control filtering thresholds, the species identified corresponded with both routine microbiological services diagnoses and MiSeq results. By analysing sequences in real time, causes of infection were robustly detected within ten minutes from initiation of sequencing. We demonstrate initial proof of concept that metagenomic MinION sequencing can provide rapid, accurate diagnosis for prosthetic joint infections. We demonstrate a novel, scalable pipeline for real-time analysis of MinION sequence data. The high proportion of human DNA in extracts prevents further genotyping, and methods to reduce this could increase genome depth and allow antimicrobial resistance profiling.