TY - JOUR
T1 - Investigation of Detection Limits and the Influence of DNA Extraction and Primer Choice on the Observed Microbial Communities in Drinking Water Samples Using 16S rRNA Gene Amplicon Sequencing
AU - Brandt, Jakob
AU - Albertsen, Mads
PY - 2018/9/7
Y1 - 2018/9/7
N2 - In recent years, 16S rRNA gene amplicon sequencing has been widely adopted for analyzing the microbial communities in drinking water (DW). However, no comprehensive attempts have been made to illuminate the inherent method biases specifically relating to DW communities. In this study, we investigated the impact of DNA extraction and primer choice on the observed microbial community, and furthermore estimated the detection limit of the 16S rRNA gene amplicon sequencing in these experimental settings. Of the two DNA extraction kits investigated, the PowerWater DNA Isolation Kit resulted in higher yield, better reproducibility and more OTUs identified compared to the FastDNA SPIN Kit for Soil, which is also commonly used within DW microbiome research. The use of three separate primer-sets targeting the V1-3, V3-4, and V4 region of the 16S rRNA gene revealed large differences in OTU abundances, with some of the primers unable to detect entire phyla. Estimations of the detection limit were based on bacteria-free water samples (1 L) spiked with Escherichia coli cells in different concentrations [10
1-10
6 cells/ml]. E. coli could be detected in all samples, however, samples with ~10
1 cells/ml had several contaminating OTUs constituting approximately 8% of the read abundances. Based on our findings, we recommend using the PowerWater DNA Isolation Kit for DNA extraction in combination with PCR amplification of the V3-4 or V4 region for DW samples if a broad overview of the microbial community is to be obtained.
AB - In recent years, 16S rRNA gene amplicon sequencing has been widely adopted for analyzing the microbial communities in drinking water (DW). However, no comprehensive attempts have been made to illuminate the inherent method biases specifically relating to DW communities. In this study, we investigated the impact of DNA extraction and primer choice on the observed microbial community, and furthermore estimated the detection limit of the 16S rRNA gene amplicon sequencing in these experimental settings. Of the two DNA extraction kits investigated, the PowerWater DNA Isolation Kit resulted in higher yield, better reproducibility and more OTUs identified compared to the FastDNA SPIN Kit for Soil, which is also commonly used within DW microbiome research. The use of three separate primer-sets targeting the V1-3, V3-4, and V4 region of the 16S rRNA gene revealed large differences in OTU abundances, with some of the primers unable to detect entire phyla. Estimations of the detection limit were based on bacteria-free water samples (1 L) spiked with Escherichia coli cells in different concentrations [10
1-10
6 cells/ml]. E. coli could be detected in all samples, however, samples with ~10
1 cells/ml had several contaminating OTUs constituting approximately 8% of the read abundances. Based on our findings, we recommend using the PowerWater DNA Isolation Kit for DNA extraction in combination with PCR amplification of the V3-4 or V4 region for DW samples if a broad overview of the microbial community is to be obtained.
KW - 16S rRNA gene amplicon sequencing
KW - DNA
KW - Detection limit
KW - Drinking water
KW - Extraction
KW - Primer
KW - Sampling
UR - http://www.scopus.com/inward/record.url?scp=85053029718&partnerID=8YFLogxK
U2 - 10.3389/fmicb.2018.02140
DO - 10.3389/fmicb.2018.02140
M3 - Journal article
C2 - 30245681
SN - 1664-302X
VL - 9
JO - Frontiers in Microbiology
JF - Frontiers in Microbiology
M1 - 2140
ER -