DNA extraction of microbial DNA directly from infected tissue: an optimized protocol for use in nanopore sequencing

Karin Helmersen,Hege Vangstein Aamot

doi:10.1038/s41598-020-59957-6

Abstract

Identification of bacteria causing tissue infections can be comprehensive and, in the cases of non- or slow-growing bacteria, near impossible with conventional methods. Performing shotgun metagenomic sequencing on bacterial DNA extracted directly from the infected tissue may improve time to diagnosis and targeted treatment considerably. However, infected tissue consists mainly of human DNA (hDNA) which hampers bacterial identification. In this proof of concept study, we present a modified version of the Ultra-Deep Microbiome Prep kit for DNA extraction procedure, removing additional human DNA. Tissue biopsies from 3 patients with orthopedic implant-related infections containing varying degrees of Staphylococcus aureus were included. Subsequent DNA shotgun metagenomic sequencing using Oxford Nanopore Technologies’ (ONT) MinION platform and ONTs EPI2ME WIMP and ARMA bioinformatic workflows for microbe and antibiotic resistance genes identification, respectively. The modified DNA extraction protocol led to an additional ~10-fold reduction of human DNA while preserving S. aureus DNA. Including the DNA sequencing and bioinformatics analyses, the presented protocol has the potential of identifying the infection-causing pathogen in infected tissue within 7 hours after biopsy. However, due to low number of S. aureus reads, positive identification of antibiotic resistance genes was not possible.

Highlights

As next-generation sequencing (NGS), with its multitude of advantages, is approaching acceptance as the gold standard in bacteriology[1], the demand for optimal DNA extraction procedures are increasing
A modified version of the Ultra-Deep Microbiome Prep kit for DNA extraction, facilitated a further ~10-fold reduction of human DNA while preserving S. aureus DNA. This resulted in increased sensitivity of shotgun metagenomic sequencing and improved pathogen identification
When using shotgun metagenomic sequencing, human DNA (hDNA) poses challenges when the human to microbe ratio is high, as it is in tissue

Summary

Introduction

As next-generation sequencing (NGS), with its multitude of advantages, is approaching acceptance as the gold standard in bacteriology[1], the demand for optimal DNA extraction procedures are increasing. Being able to extract microbial DNA directly from human samples followed by shotgun metagenomic sequencing, where all DNA in a complex sample is identified, can reduce time to diagnosis and targeted treatment. This is especially important in cases with slow-growing or difficult to cultivate microbes. The NGS sequencing platforms widely used for metagenomic sequencing, such as Ion Torrent and Illumina, require comprehensive pre-sequencing preparation of samples and require the sequencing run to completion before analysis can start (the exception being a recently described method that can analyze raw Illumina data before run completion (LiveKraken[5]) These obstacles may be overcome by using nanopore sequencing technology (Oxford Nanopore Technologies (ONT), Oxford, UK) where pre-sequencing preparation is short (15 min–2 h depending on DNA concentration) and data analysis can be done in near real-time using the web-based EPI2ME bioinformatics. The expeditious identification of these infective agents is of major importance for patient treatment and could improve outcome

Methods

Results

Conclusion