The effect of DNA extraction methodology on gut microbiota research applications

Abstract

BackgroundThe effect that traditional and modern DNA extraction methods have on applications to study the role of gut microbiota in health and disease is a topic of current interest. Genomic DNA was extracted from three faecal samples and one probiotic capsule using three popular methods; chaotropic (CHAO) method, phenol/chloroform (PHEC) extraction, proprietary kit (QIAG). The performance of each of these methods on DNA yield and quality, microbiota composition using quantitative PCR, deep sequencing of the 16S rRNA gene, and sequencing analysis pipeline was evaluated.ResultsThe CHAO yielded the highest and the QIAG kit the lowest amount of double-stranded DNA, but the purity of isolated nucleic acids was better for the latter method. The CHAO method yielded a higher concentration of bacterial taxa per mass (g) of faeces. Sequencing coverage was higher in CHAO method but a higher proportion of the initial sequencing reads were retained for assignments to operational taxonomic unit (OTU) in the QIAG kit compared to the other methods. The QIAG kit appeared to have longer trimmed reads and shorter regions of worse quality than the other two methods. A distinct separation of α-diversity indices between different DNA extraction methods was not observed. When compositional dissimilarities between samples were explored, a strong separation was observed according to sample type. The effect of the extraction method was either marginal (Bray–Curtis distance) or none (unweighted Unifrac distance). Taxon membership and abundance in each sample was independent of the DNA extraction method used.ConclusionsWe have benchmarked several DNA extraction methods commonly used in gut microbiota research and their differences depended on the downstream applications intended for use. Caution should be paid when the intention is to pool and analyse samples or data from studies which have used different DNA extraction methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2171-7) contains supplementary material, which is available to authorized users.

Robust Microbiota-Based Diagnostics for Inflammatory Bowel Disease

“…Our replicate data sets were limited to only two different platforms and two hospitals, and the betweenhospital replicate data set was an unbalanced subset of the original cohort and rather small. Finally, since DNA extraction has been shown to be an important factor in microbiota composition variation (28,29), our replicate data sets did not include reisolation of the DNA, rather than reprocessing of the same DNA isolates. The DNA isolates were therefore stored until the two replicates were analyzed, which may also introduce some degree of variation.…”

Shotgun metagenomics, from sampling to analysis

“…However, it is difficult to mimic the complexity of real microbial communities using simplified mocks, and impossible to test for the efficiency of the extraction step for unknown/uncultured organisms. Much evidence suggests that incorporating a bead-beating step into the DNA extraction process improves yield and representativeness of resulting species profiles compared to chemical-only lysis 31,135 (ref#133 C.Q.,N.J.L.). However, this type of approach does typically result in more sheared DNA, potentially limiting the power of burgeoning long read sequencing technologies.…”

Effect of the extraction and purification of soilDNAand pooling ofPCRamplification products on the description of bacterial and archaeal communities

“…Many studies have shown that using distinct DNA extraction methods may result in very different band patterns. Recently, studies using next-generation sequencing techniques to compare the use of different protocols for DNA extraction from soil (Feinstein et al 2009;Terrat et al 2012;Vishnivetskaya et al 2014;W€ ust et al 2016), marine sediment (Cruaud et al 2014), human gut (Kennedy et al 2014b;Gerasimidis et al 2016), rumen (Henderson et al 2013;Fliegerova et al 2014) and activated sludge samples (Guo and Zhang 2013) also suggest that different extraction methods are more efficient in extracting the DNA of different microbial groups. For example, the abundance of Acidobacteria, Proteobacteria, Actinobacteria and Firmicutes in soil was underestimated or overestimated depending on the DNA extraction method (Terrat et al 2012).…”