Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis

Abstract

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.

Application Progress of High-Throughput Sequencing in Ocular Diseases

“…With the advancement of HTS technology, we are gaining increasing information on genes, transcripts, and non-coding RNAs (such as miRNA) [ 43 , 59 , 62 ]. The recent full-length nanopore 16S sequencing technology has the advantages of portability, low cost, and rapid sequencing [ 84 ]. It is increasingly used in the etiological diagnosis of various eye diseases and has also contributed greatly to exploring the normal microbiota that maintains ocular surface homeostasis [ 39 , 42 ].…”

16S rRNA nanopore sequencing for the diagnosis of ocular infection: a feasibility study

“…Low et al reported 16S rRNA gene analysis using a portable nanopore sequencer in two cases of keratitis 30. Jun et al also used a nanopore sequencer in eight cases of endophthalmitis 31.…”

“…Low et al reported 16S rRNA gene analysis using a portable nanopore sequencer in two cases of keratitis. 30 Jun et al also used a nanopore sequencer in eight cases of endophthalmitis. 31 Low et al reported the advantages of gene analysis using MinIONin detecting causative organisms after administering antimicrobial eye drops.…”

Contact Lens-Associated Infectious Keratitis: Update on Diagnosis and Therapy

“…The 16S rRNA gene sequence is 1,550 bp long, and it is composed of nine variable regions (V1-V9) interspaced in more conserved regions. By amplifying the 16S rRNA region with PCR, the background host contamination encountered in routing culturing techniques is reduced significantly [98].…”

“…Second-generation platforms (Illumina HiSeq 2500), although able to generate high sequence throughput data in a single reaction, they only sequenced part of the 16S gene [94,103,104]. Current third-generation platforms use nanopore sequencing technology directly from clinical samples to diagnose bacterial keratitis in real time and with higher accuracy [98].…”