Comparison of Illumina Versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota

Abstract

Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME and an in house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 and R9.2 were compared.

Nanopore Sequencing Provides Rapid and Reliable Insight Into Microbial Profiles of Intensive Care Units

“…Literature shows that the use of Oxford Nanopore's commercial barcoding primers also potentially interfere with one's capacity to detect certain bacterial taxa in complex samples. Corynebacterium, Pseudomonas and Bifidobacterium have been reported to be some of the affected genera (9,10,25).…”

“…Moreover, size range cutoffs chosen to filter reads certainly have an impact on the number of reads retrieved in a given experiment. Thus, most reports for microbial community analysis using 16S we found applied different Q Score thresholds (usually Q Score > 7) and variable cutoff values (e.g., 1,300-1,950 bp, <1,700 bp, 1,400-1,700 bp, and 1,350-1,650 bp) (9,10,25,27).…”

“…While promising, 16S-based species-level microbial profiling using nanopore remains a rather controversial topic. Current error rates reported for long-read basecalling algorithms are usually higher than the differences between closely related species, which makes species-level assignment unadvisable due to the possibility of false positives (9,10). Using a mock community, Winand and collaborators reported that, even though genuslevel classification of nanopore is highly accurate, the use of absolute number of reads as a "tiebreak" between organisms of the same genus would not reflect the real composition of the community for all present species (9).…”

“…Despite gaining traction, nanopore sequencing protocols and analysis pipelines are still being validated by the community at large, and many research groups have been thoroughly evaluating its performance against traditional (usually Illumina) platforms (9)(10)(11)(12). In the present study, we investigated how nanoporebased long-read sequencing compares to Illumina sequencing for the study of complex microbial communities from hospital surfaces.…”

Fecal genomic DNA extraction method impacts outcome of MinION based metagenome profile of tuberculosis patients

“…And MinION and Illumina platforms generated comparable results from different biofluids including human nasal samples. 25 Many metagenomics studies adopt commercial kits for DNA extraction and reports demonstrated that manual method processed fecal samples better captures the microbial diversity. 26 In this study we compared two manual methods of DNA extraction for further processing.…”