Beyond 16S sequencing for microbiome research: Nanopore sequencing resource at The University of Toledo Microbiome Consortium

Abstract

Objective: The recognition that humans are holobionts has opened a new research area to examine the contributions of the microbiome to health and disease. This requires a full understanding of the identities of microbiota at the taxonomical level of the species. Introduction: The University of Toledo Microbiome Consortium currently offers the second-generation Illumina sequencing platform for 16S sequencing to determine microbiota composition. 16S sequencing of variable regions of the 16S operon, such as V3, V4, and the combination of V3 to V4, has long been the main stay for analyzing the composition of microbiota. The advantage of this approach is the ability to screen large numbers of samples at a lower cost per sample. However, the major drawback of this approach is a lower resolution whereby taxonomical identities of microbiota are limited to the genus level. To overcome this disadvantage, we introduced the third-generation sequencing technology from Nanopore into our pipeline. The known advantages of this approach are longer reads and the ability to sequence DNA in the native form without PCR-induced bias. Here we sought to conduct a comparative study of the relative effciencies of the 16S versus the nanopore technology for metagenomics. Methods: Genomic DNA was isolated from control and experimental rat fecal samples provided by an end-user. Libraries were prepared using Ligation sequencing DNA V14 (SqK-LSK114) on a Flow cell R10.4.1and Lambda DNA was used as a sequencing control. The native Libraries were run for 72hr, and high accuracy base calling was performed. The Fastq files (passed) generated by GridION were run on cloud-based CZ-ID pipeline with host and human read filtering, the results were normalized to one million bases sequenced from reads of each sample. Results and Conclusions: The 16S sequencing data highlighted the relative abundance of different taxa up to genus level such as Lactobacillus and Erysipelatoclostridium in the experimental group where as Stepcococcus and prevotellaceaeGa6A1group in the control group. However, Nanopore sequencing further identified twenty different species of Lactobacillus and , four different species of Erysipelatoclostridium. Similarly, nanopore sequencing was able to profile diverse species belonging to genera prevotellaceaeGa6A1group and Streptococcus. This species level resolution will greatly help in focusing on specific species and the pathways that a researcher intends to pursue. Therefore, the nanopore sequencing presents an opportunity to explore sequencing with a small footprint in terms of instrumentation and associated costs that greatly helps even a smaller laboratory to adopt and reap the benefits of species level resolution of metagenomic data. Summary: This study correlated well with 16S sequencing results at the genus level, while offering the added benefit of species level Identification and classification. Authors acknowledge the National Institutes of Health for funding support to Bina Joe (R01HL1430820). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.