Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis
Background Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-tim...
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PeerJ Inc.
2021-02-01
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| author | Liying Low Pablo Fuentes-Utrilla James Hodson John D. O’Neil Amanda E. Rossiter Ghazala Begum Kusy Suleiman Philip I. Murray Graham R. Wallace Nicholas J. Loman Saaeha Rauz West Midlands Collaborative Ophthalmology Network for Clinical Effectiveness & Research by Trainees (WM CONCERT) |
| author_facet | Liying Low Pablo Fuentes-Utrilla James Hodson John D. O’Neil Amanda E. Rossiter Ghazala Begum Kusy Suleiman Philip I. Murray Graham R. Wallace Nicholas J. Loman Saaeha Rauz West Midlands Collaborative Ophthalmology Network for Clinical Effectiveness & Research by Trainees (WM CONCERT) |
| author_sort | Liying Low |
| collection | DOAJ |
| description | Background Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis. Methods Using full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods. Results We found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results. Conclusion We have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing. |
| first_indexed | 2024-03-09T07:07:41Z |
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| language | English |
| last_indexed | 2024-03-09T07:07:41Z |
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| spelling | doaj.art-bfd1b38957d346f69decf2549dcaa8892023-12-03T09:28:24ZengPeerJ Inc.PeerJ2167-83592021-02-019e1077810.7717/peerj.10778Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitisLiying Low0Pablo Fuentes-Utrilla1James Hodson2John D. O’Neil3Amanda E. Rossiter4Ghazala Begum5Kusy Suleiman6Philip I. Murray7Graham R. Wallace8Nicholas J. Loman9Saaeha Rauz10West Midlands Collaborative Ophthalmology Network for Clinical Effectiveness & Research by Trainees (WM CONCERT)11Academic Unit of Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, West Midlands, UKMicrobesNG/School of Biosciences, University of Birmingham, Birmingham, West Midlands, UKQueen Elizabeth Hospital, University Hospitals Birmingham NHS Foundation Trust, Birmingham, West Midlands, UKInstitute of Inflammation and Ageing, University of Birmingham, Birmingham, West Midlands, UKInstitute of Microbiology and Infection, University of Birmingham, Birmingham, West Midlands, UKInstitute of Inflammation and Ageing, University of Birmingham, Birmingham, West Midlands, UKAcademic Unit of Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, West Midlands, UKAcademic Unit of Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, West Midlands, UKAcademic Unit of Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, West Midlands, UKMicrobesNG/School of Biosciences, University of Birmingham, Birmingham, West Midlands, UKAcademic Unit of Ophthalmology, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, West Midlands, UKBirmingham and Midland Eye Centre, Sandwell and West Birmingham Hospitals National Health Service (NHS) Trust, Birmingham, West Midlands, UKBackground Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis. Methods Using full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods. Results We found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results. Conclusion We have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.https://peerj.com/articles/10778.pdfNanopore sequencingEye infectionMicrobial keratitisFull length 16S rRNA sequencingCornea infectionEye swab |
| spellingShingle | Liying Low Pablo Fuentes-Utrilla James Hodson John D. O’Neil Amanda E. Rossiter Ghazala Begum Kusy Suleiman Philip I. Murray Graham R. Wallace Nicholas J. Loman Saaeha Rauz West Midlands Collaborative Ophthalmology Network for Clinical Effectiveness & Research by Trainees (WM CONCERT) Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis PeerJ Nanopore sequencing Eye infection Microbial keratitis Full length 16S rRNA sequencing Cornea infection Eye swab |
| title | Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis |
| title_full | Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis |
| title_fullStr | Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis |
| title_full_unstemmed | Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis |
| title_short | Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis |
| title_sort | evaluation of full length nanopore 16s sequencing for detection of pathogens in microbial keratitis |
| topic | Nanopore sequencing Eye infection Microbial keratitis Full length 16S rRNA sequencing Cornea infection Eye swab |
| url | https://peerj.com/articles/10778.pdf |
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