Microbiome

Beyond the hypervariable regions: Full-length 16S Oxford Nanopore sequencing of rumen microbiome samples to get the most out of a metagenomic sample

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Abstract

The objective of this study was to identify an optimal method to sequence the full-length 16S rRNA gene for bacterial classification of complex metagenomic samples. Fifty nanogram samples of DNA extracted from rumen liquid collected via canula from Jersey and Holstein cows were amplified using primers (27F and 1492R) designed to amplify the full-length 16S rRNA gene. The resulting amplicon (unpurified) or gel-purified amplicon samples were sequenced using the Nanopore Rapid Barcoding Kit (RBC) or the Native Barcoding Kit (NBC). Barcoded libraries were sequenced via PromethION flow cells parameterized to discard reads outside 1000-3000 bp or with a Q-score of < 15. Reads were quality filtered and cleaned (PoreChop) before being taxonomically classified (Kaiju) and summary statistics were generated (NanoComp). Paired t-tests were performed on within sample for NBC and RBC, while an unpaired t-test was performed on the difference in NBC and RBC for unpurified amplicon samples and gel-purified amplicon samples. A Wilcox-test was performed on classification data to identify statistical differences in taxonomic richness. The NBC produced longer mean read lengths (p< 0.0001), while the RBC produced higher quality reads (p< 0. 0001). Overall, quality metrics such as mean and median read length, mean and median quality, and number of reads were superior from unpurified amplicon compared to gel-purified amplicon (p< 0.01) samples with these differences being more pronounced in the NBC over the RBC. Mean Q-score for all samples ranged from 18.0 to 19.5, while the median Q-score for all samples ranged from 17.9 to 20.9. Unpurified amplicon RBC samples had the greatest taxonomic richness. Gel-purified samples had fewer unclassified reads, 0.1% and 1.0%, than unpurified samples, 2.5% and 6.6%, for NBC and RBC respectively. In conclusion, NBC provides the most reliable and accurate sequence data. Unpurified amplicon samples performed well with both sequencing protocols. While unclassified reads occurred at a higher rate with unpurified amplicon samples, their highest rate was still relatively low (RBC: 6.6%) and in silico removal of these reads is straightforward. This methodology will help further characterize the bacterial portion of the rumen microbiome though development of rapid and reproducible methods. While DNA from rumen liquids provide a large amount of DNA from each sample, this method is also compatible with oral swabs, which provide less DNA, but are less invasive.

Keywords: 2026

How to Cite:

Amorin De Hegedus, R., Beskid, J., Costello, M., McClure, J., Panke-Buisse, K. & Young, J., (2026) “Beyond the hypervariable regions: Full-length 16S Oxford Nanopore sequencing of rumen microbiome samples to get the most out of a metagenomic sample”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2294620. doi: https://doi.org/10.31274/wcgalp.24324

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