Comprehensive Wet-Bench and Bioinformatics Workflow for Complex Microbiota Using Oxford Nanopore Technologies
Christoph Ammer‐Herrmenau, Nina Pfisterer, Tim van den Berg, Ivana Gavrilova, Ahmad Amanzada, Shiv K. Singh, Alaa Khalil, Rohia Alili, Eugeni Belda, Karine Clément, Ahmed Abd El Wahed, ElSagad Eltayeb Gady, Martin Haubrock, Tim Beißbarth, Volker Ellenrieder, Albrecht Neeße
Abstract
Advanced microbiome analysis relies on sequencing of short DNA fragments from microorganisms like bacteria, fungi, and viruses. More recently, long fragment DNA sequencing of 3rd generation sequencing has gained increasing importance and can be rapidly conducted within a few hours due to its potential real-time sequencing. However, the analysis and correct identification of the microbiome relies on a multitude of factors, such as the method of sampling, DNA extraction, sequencing, and bioinformatic analysis. Scientists have used different protocols in the past that do not allow us to compare results across different studies and research fields. Here, we provide a comprehensive workflow from DNA extraction, sequencing, and bioinformatic workflow that allows rapid and accurate analysis of human buccal and rectal swabs with reproducible protocols. This workflow can be readily applied by many scientists from various research fields that aim to use long-fragment microbiome sequencing.