Biases from different DNA extraction methods in intestine microbiome research based on 16S rDNA sequencing: a case in the koi carp, Cyprinus carpio var. Koi

Abstract

This study examined the technical bias associated with different DNA extraction methods used in microbiome research. Three methods were used to extract genomic DNA from the same intestinal microbiota sample that was taken from the koi carp Cyprinus carpio var. koi, after which their microbial diversity and community structure were investigated on the basis of a 16S rDNA high-throughput sequencing analysis. Biased results were observed in relation to the number of reads, alpha diversity indexes and taxonomic composition among the three DNA extraction protocols. A total of 1,381 OTUs from the intestinal bacteria were obtained, with 852, 759, and 698 OTUs acquired, using the Lysozyme and Ultrasonic Lysis method, Zirmil-beating Cell Disruption method, and a QIAamp Fast DNA Stool Mini Kit, respectively. Additionally, 336 OTUs were commonly acquired, using the three methods. The results showed that the alpha diversity indexes (Rarefaction, Shannon, and Chao1) of the community that were determined using the Lysozyme and Ultrasonic Lysis method were higher than those obtained with the Zirmil-beating Cell Disruption method, while the Zirmil method results were higher than those measured, using the QIAamp Fast DNA Stool Mini Kit. Moreover, all the major phyla (ratio>1%) could be identified with all three DNA extraction methods, but the phyla present at a lower abundance (ratio <1%) could not. Similar findings were observed at the genus level. Taken together, these findings indicated that the bias observed in the results about the community structure occurred primarily in OTUs with a lower abundance. The results of this study demonstrate that possible bias exists in community analyses, and researchers should therefore be conservative when drawing conclusions about community structures based on the currently available DNA extraction methods.

Community composition and development of the post-weaning piglet gut microbiome

“…Systematic taxonomic bias in microbiome studies, resulting from differences in cell wall structures between Gram positive and Gram negative bacteria, have previously been reported; sample treatment with enzymatic cocktails can modestly reduce this bias [102][103][104] . Although we implemented this step in our work ow, it seems that, from the read abundance of our mock community, which contained three Gram negative and four Gram positive strains, a bias towards Gram negative taxa may still be present.…”

“…were performed. Alpha-diversity and beta-diversity were analyzed and the results were visualized with R 108 and R packages [91][92][93][94][95][96][97][98][99][100][101][102][103][104][105][106][107] . The data analysis is schematically represented in Supplementary Fig.…”

Multicenter assessment of microbial community profiling using 16S rRNA gene sequencing and shotgun metagenomic sequencing

“…Previous studies have shown that different DNA extraction approaches (kits) vary in efficiency for different types of microbes, especially for hard-to-lyse microbes (such as G+ bacteria), leading to significant deviations in DNA yield and bacterial composition [40] , [41] , [42] . This issue is ubiquitous in the laboratories participating in this evaluation study.…”

Phylogenetic diversity analysis of shotgun metagenomic reads describes gut microbiome development and treatment effects in the post-weaned pig

“…Beta diversity analysis revealed a distinct and consistent change of the microbial The following taxa were responsible for variation throughout the trial: Clostridiales The effect of time was also clear in alpha diversity, where all the piglet cohorts followed a similar trend over time, independent of the treatment (S23; Supplementary file 5). Upon arrival on the trial site, the piglets' unrooted PD was lower than the sows (sows: 125…”

“…This is not the case for real-world samples where mapping against a database (which completeness relies on studied and often cultivable organisms) would narrow the view on the true diversity within the sample.Positive controls with well-studied members and known ratios within the samples, has proven to be a valuable tool to assess consistency among technical replicates across batches and to detect possible biases derived from the DNA extraction method. Systematic taxonomic bias in microbiome studies, resulting from differences in cell wall structures between Gram positive and Gram negative bacteria, have previously been reported; sample treatment with enzymatic cocktails can modestly reduce this bias[124][125][126] .…”