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Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis.

ABSTRACT: BACKGROUND: In recent years, studies on the human intestinal microbiota have attracted tremendous attention. Application of next generation sequencing for mapping of bacterial phylogeny and function has opened new doors to this field of research. However, little attention has been given to the effects of choice of methodology on the output resulting from such studies. RESULTS: IN THIS STUDY WE CONDUCTED A SYSTEMATIC COMPARISON OF THE DNA EXTRACTION METHODS USED BY THE TWO MAJOR COLLABORATIVE EFFORTS: The European MetaHIT and the American Human Microbiome Project (HMP). Additionally, effects of homogenizing the samples before extraction were addressed. We observed significant differences in distribution of bacterial taxa depending on the method. While eukaryotic DNA was most efficiently extracted by the MetaHIT protocol, DNA from bacteria within the Bacteroidetes phylum was most efficiently extracted by the HMP protocol. CONCLUSIONS: Whereas it is comforting that the inter-individual variation clearly exceeded the variation resulting from choice of extraction method, our data highlight the challenge of comparing data across studies applying different methodologies.

SUBMITTER: Wesolowska-Andersen A

PROVIDER: S-EPMC4063427 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis.

Wesolowska-Andersen Agata A Bahl Martin Iain MI Carvalho Vera V Kristiansen Karsten K Sicheritz-Pontén Thomas T Gupta Ramneek R Licht Tine Rask TR

Microbiome 20140605

<h4>Background</h4>In recent years, studies on the human intestinal microbiota have attracted tremendous attention. Application of next generation sequencing for mapping of bacterial phylogeny and function has opened new doors to this field of research. However, little attention has been given to the effects of choice of methodology on the output resulting from such studies.<h4>Results</h4>IN THIS STUDY WE CONDUCTED A SYSTEMATIC COMPARISON OF THE DNA EXTRACTION METHODS USED BY THE TWO MAJOR COLL ...[more]

PMID: 24949196

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Project description:Understanding the unique and unexplored microbial environment of xylem sap is starting to be of relevant importance for plant health, as it could include microbes that may protect plants against xylem-limited pathogens, such as Verticillium dahliae and Xylella fastidiosa. In this study, we evaluated the effects that the method for extracting the xylem bacterial communities, the plant age and the PCR primers may have on characterizing the xylem-bacterial-community composition by using an NGS approach. Xylem sap was extracted from xylem vessels by using a Scholander pressure chamber, or by macerating wood shavings that were obtained from xylem tissues by using branches from 10-year-old olive trees, or the entire canopy of 1-year-old olive plantlets. Additionally, we compared four different PCR-primer pairs that target 16S rRNA for their efficacy to avoid the coamplification of mitochondria and chloroplast 16S rRNA, as this represents an important drawback in metabarcoding studies. The highest amplifications in the mitochondria and chloroplast reads were obtained when using xylem woody chips with the PCR1-799F/1062R (76.05%) and PCR3-967F/1391R (99.96%) primer pairs. To the contrary, the PCR2-799F/1115R and PCR4-799F/1193R primer pairs showed the lowest mitochondria 16S rRNA amplification (<27.48%), no chloroplast sequences and the highest numbers of bacterial OTUs identified (i.e., 254 and 266, respectively). Interestingly, only 73 out of 172 and 46 out of 181 genera were shared between the xylem sap and woody chips after amplification with PCR2 or PCR4 primers, respectively, which indicates a strong bias of the bacterial-community description, depending on the primers used. Globally, the most abundant bacterial genera (>60% of reads) included Anoxybacillus, Cutibacterium, Pseudomonas, Spirosoma, Methylobacterium-Methylorubrum and Sphingomonas; however, their relative importance varied, depending on the matrix that was used for the DNA extraction and the primer pairs that were used, with the lowest effect due to plant age. These results will help to optimize the analysis of xylem-inhabiting bacteria, depending on whether whole xylematic tissue or xylem sap is used for the DNA extraction. More importantly, it will help to better understand the driving and modifying factors that shape the olive-xylem-bacterial-community composition.

Project description:BackgroundSequence-based methods for the detection of bacteria such as 16S rRNA amplicon sequencing and metagenomics can provide a comprehensive view of the bacterial microbiome of food. These methods rely on the detection of gene sequences to indicate the presence of viable bacteria. This indirect form of detection can be prone to experimental artefacts. Sample handling and processing are key sources of variation that require standard approaches. Extracting sufficient quantities of high quality DNA from food matrices is challenging because target bacterial species are usually minor components of the microbiota and foods contain an array of compounds that are inhibitory to downstream DNA applications. Here, three DNA extraction methods are compared for their ability to extract high quality bacterial DNA from retail chicken breast rinses, with or without enrichment. Method performance was assessed by comparing ease of use, DNA yield, DNA quality, PCR amplicon yield, and the detection of bacterial taxa by 16S rRNA amplicon sequencing.ResultsAll three DNA extraction methods yielded DNA of sufficient quantity and quality to perform quantitative PCR and 16S rRNA amplicon sequencing. The extraction methods differed in ease of use, with the two commercial kits (PowerFood, PowerSoil) offering considerable time and cost savings over a hybrid method that used laboratory reagents for lysis and commercial column based kits for further purification. Bacterial richness as determined by 16S rRNA amplicon sequencing was similar across the three DNA extraction methods. However, differences were noted in the relative abundance of bacterial taxa, with significantly higher abundance of Gram-positive genera detected in the DNA samples prepared using the PowerFood DNA extraction kit.ConclusionThe choice of DNA extraction method can affect the detection of bacterial taxa by 16S rRNA amplicon sequencing in chicken meat rinses. Investigators should be aware of this procedural bias and select methods that are fit for the purposes of their investigation.

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Dataset Information

Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis.

Publications

Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis.

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