Benchmarking DNA isolation kits used in analyses of the urinary microbiome

Lisa Karstens,Tatyana A Sysoeva,Cindy L Amundsen,Nazema Y Siddiqui,Alecsander Barstad,Tamara Zaza

doi:10.1038/s41598-021-85482-1

Abstract

The urinary microbiome has been increasingly characterized using next-generation sequencing. However, many of the technical methods have not yet been specifically optimized for urine. We sought to compare the performance of several DNA isolation kits used in urinary microbiome studies. A total of 11 voided urine samples and one buffer control were divided into 5 equal aliquots and processed in parallel using five commercial DNA isolation kits. DNA was quantified and the V4 segment of the 16S rRNA gene was sequenced. Data were processed to identify the microbial composition and to assess alpha and beta diversity of the samples. Tested DNA isolation kits result in significantly different DNA yields from urine samples. DNA extracted with the Qiagen Biostic Bacteremia and DNeasy Blood & Tissue kits showed the fewest technical issues in downstream analyses, with the DNeasy Blood & Tissue kit also demonstrating the highest DNA yield. Nevertheless, all five kits provided good quality DNA for high throughput sequencing with non-significant differences in the number of reads recovered, alpha, or beta diversity.

Highlights

In addition to sample collection, handling and storage, the DNA isolation methods used are another important step for microbiome analysis where bias could be introduced prior to sequencing
The total DNA concentration recovered from each DNA isolation kit was highly variable (Fig. 1A, Table S3) with the Qiagen DNeasy Blood and Tissue kit resulting in the highest concentrations and the Promega kit with the lowest (Kruskal–Wallis p = 0.0007 overall, pairwise Wilcoxon rank sum < 0.05)
Though many of our downstream assessments showed non-significant differences, our data do not support the assumption that all DNA isolation kits perform in urinary microbiome studies, as we identified some qualitative differences in recovery of Gram positive versus Gram negative organisms, and some differences in overall performance with low biomass samples

Summary

Introduction

In addition to sample collection, handling and storage, the DNA isolation methods used are another important step for microbiome analysis where bias could be introduced prior to sequencing At this step, human and microbial DNA are extracted from the proteins, salts, and other components of the physiologic sample. Many commercial kits and custom protocols for DNA isolation were developed for microbiome analyses for microbe-rich or microbe-poor e nvironments[19,22,25,26] This tailoring of DNA isolation methods was required to achieve more representative identification and quantification of the microbial composition for each respective environment. As there are no studies directly comparing the results obtained when urine samples are subjected to different commercial DNA isolation kits, our primary objective was to assess whether recovered microbes identified by 16S rRNA sequencing differ based on the DNA isolation protocol

Objectives

Methods

Results

Discussion

Conclusion