Identification and removal of contaminating microbial DNA from PCR reagents: impact on low-biomass microbiome analyses

Abstract

Significance and Impact of the Study: Reagent contamination with microbial DNA is a major problem in microbiome studies of low microbial biomass samples. Levels of such contaminating DNA often outweigh what is present in the sample and heavily confound subsequent data analysis. Previous studies have suggested this contamination is primarily derived from DNA extraction kits. Here, we identified the PCR master mix as the primary source of contamination, and showed that enzymatic removal of the contamination drastically reduced the blank signal and improved precision. Decontamination of PCR master mixes may have the potential to improve the sensitivity and accuracy of low-biomass microbiome studies. AbstractReagent-derived contamination can compromise the integrity of microbiome data, particularly in low microbial biomass samples. This contamination has recently been attributed to the 'kitome' (contamination introduced by the DNA extraction kit), prior to which attention was mostly paid to potential contamination introduced by PCR reagents. In this study, we assessed the proportion to which our DNA extraction kit and PCR master mix introduce contaminating microbial DNA to bacterial microbial profiles generated by 16S rRNA gene sequencing. Utilizing a commercial dsDNase treatment protocol to decontaminate the PCR master mix, we demonstrated that the vast majority of contaminating DNA was derived from the PCR master mix. Importantly, this contamination was almost completely eliminated using the simple dsDNase treatment, resulting in a 99% reduction in contaminating bacterial reads. We suggest that dsDNase treatment of PCR reagents should be explored as a simple and effective way of reducing contamination in low-biomass microbiome studies and producing more robust and reliable data.Letters in Applied Microbiology 68, 2--8

Comparison of Bacterial DNA Profiles in Mid-Trimester Amniotic Fluid Samples From Preterm and Term Deliveries

“…The V6 region of the 16S rRNA gene was amplified in 20 µL reactions containing 6 µL of template or water (negative template control), 1× TaqMan Fast Advanced Master Mix (Applied Biosystems), 0.9 µM each of the forward (917F 5 -GAATTGACGGGGRCCCGC-3 ) and reverse (1033R 5 -TGCGGGACTTAACCCAACA-3 ) primers, 0.25 µM of probe (5 -FAM-CACGAGCTGACGACARCCATGCA-TAMRA-3 ), and 0.95 µL of water. Master mix solutions were treated with a PCR Decontamination Kit (ArcticZymes R ), which consisted of a double-stranded DNase (dsDNase) and DTT (which helps to inactivate the dsDNase), as previously described (Stinson et al, 2018). Briefly, master mix solutions (including primers and probes) were treated with 0.5 µl of dsDNase and 0.5 µl of DTT per 20 µl reaction, then incubated at 37 • C for 20 min (dsDNase activation), followed by incubation at 60 • C for 20 min (dsDNase inactivation).…”

“…Unfortunately, most previous studies are complicated by the fact that appropriate and stringent extraction and PCR controls were not used. None of these studies attempted to eliminate reagent-derived contamination using laboratorybased techniques, such as those previously described by our group (Stinson et al, 2018). This makes it difficult to make conclusions on the true level of bacterial DNA in their samples.…”

Revealing the secret life of skin ‐ with the microbiome you never walk alone

“…Both 16S rRNA gene sequencing and shotgun metagenomic sequencing require attention to contaminations. Contaminating DNA coming from DNA extraction reagents, kits (the so-called kitome) [86] and water has been reported in several studies [87][88][89][90][91].…”

Total mRNA Quantification in Single Cells: Sarcoma Cell Heterogeneity

“…Instead, cDNA was generated from nucleic acids in the reverse transcription step [40]. Most reverse transcriptases and DNA polymerases contain nucleic acid residues from the enzyme production that is amplifiable [41]. However, in our data, this background noise was not relevant, since the amount of preamplified cDNA in cell-well negatives was several times lower compared to the amount of cDNA in single cells.…”