Impact of sequence variant detection and bacterial DNA extraction methods on the measurement of microbial community composition in human stool

Abstract

BackgroundThe human gut microbiome has been widely studied in the context of human health and metabolism, however the question of how to analyze this community remains contentious. This study compares new and previously well established methods aimed at reducing bias in bioinformatics analysis (QIIME 1 and DADA2) and bacterial DNA extraction of human fecal samples in 16S rRNA marker gene surveys.ResultsAnalysis of a mock DNA community using DADA2 identified more chimeras (QIIME 1: 0.70% of total reads vs DADA2: 1.96%), fewer sequence variants, (QIIME 1: 1297.4 + 98.88 vs. DADA2: 136.27 + 11.35, mean + SD) and correct taxa at a higher resolution of classification (i.e. genus-level) than open reference OTU picking in QIIME 1. Additionally, the extraction of whole cell mock community bacterial DNA using four commercially available kits resulted in varying DNA yield, quality and bacterial community composition. Of the four kits compared, ZymoBIOMICS DNA Miniprep Kit provided the greatest yield, with a slight enrichment of Enterococcus. However, QIAamp Fast DNA Stool Mini Kit resulted in the highest DNA quality. Mo Bio PowerFecal DNA Kit had the most dramatic effect on the mock community composition, resulting in an increased proportion of members of the family Enterobacteriaceae and genus Eshcerichia as well as members of genera Lactobacillus and Pseudomonas. The presence of a sterile fecal matrix had a slight, but inconsistent effect on the yield, quality and taxa identified after extraction with all four DNA extraction kits. Extraction of bacterial DNA from native stool samples revealed a distinct effect of the DNA stabilization reagent DNA/RNA Shield on community composition, causing an increase in the detected abundance of members of orders Bifidobacteriales, Bacteroidales, Turicibacterales, Clostridiales and Enterobacteriales.ConclusionThese results confirm that the DADA2 algorithm is superior to sequence clustering by similarity to determine microbial community structure. Additionally, commercially available kits used for bacterial DNA extraction from fecal samples have some effect on the proportion of high abundance members detected in a microbial community, but it is less significant than the effect of using DNA stabilization reagent, DNA/RNA Shield.

Evaluating intraspecific genetic diversity of a fish population using environmental DNA: An approach to distinguish true haplotypes from erroneous sequences

“…In addition, both of ASV methods detected all the true haplotype with 15/15 detection rate. The great performance of the ASV methods for eliminating false positive haplotypes is consistent with previous studies that identified microorganisms from mock community samples at fine taxonomical resolutions (Callahan et al 2016;Hughes et al 2017;Kopylova et al 2016). In previous studies, the detection of true/false haplotypes relied on known reference sequences , Parsons et al 2018, while the ASV-based approach used in the present study allowed us to distinguish true haplotypes from false positive haplotypes without the reference database.…”

“…The use of high-fidelity DNA polymerase in PCR contributes to decreased errors in PCR products (Ramachandran et al 2011), but it does not completely prevent the errors. The OTU methods involve clustering of sequences that are less different from each other than a fixed similarity threshold (typically 97%; Callahan et al 2016;Hughes et al 2017). Thus, true haplotypes that are similar to each other are clustered into a single OTU, leading to incorrect evaluations of intraspecific genetic diversity.…”

Fecal storage condition induces variations of microbial composition and differential interpretation of metagenomic analysis

“…We obtained sequence reads for each sample with no differences in read counts between the two storage conditions. To join the sequence reads and ilter low-quality reads, DADA2 was used through QIIME2, which is a fast and accurate tool for metagenomic analyses [21,35,36]. We observed a difference in the number of paired-end reads, suggesting that different storage conditions could yield different microbial information.…”

Elucidating biofilm diversity on water lily leaves through 16S rRNA amplicon analysis: Comparison of four DNA extraction kits

“…Whereas the depiction of the frequencies of Gram‐negative bacteria was accurate among the four kits, there was some overestimation, especially with the strains of Bacillus and Lactobacillus . Previous studies (Yuan et al, 2012; Hughes et al, 2017; Ojo‐Okunola et al, 2020; Yang et al, 2020) had also observed a considerable overestimation of Lactobacillus sp. and Bacillus sp.…”