Dataset Information

Comparison of four DNA extraction kits efficiency for 16SrDNA microbiota profiling of diverse human samples.

ABSTRACT:

Aim

The current study investigated the performance of 4 widely used DNA extraction kits using different types of high (stool) and low biomass samples (chyme, broncho alveolar lavage and sputum).

Methods

Qiagen Powerfecal Pro DNA kit, Macherey Nucleospin Soil kit, Macherey Nucleospin Tissue Kit and MagnaPure LC DNA isolation kit III were evaluated in terms of DNA quantity, quality, diversity and composition profiles.

Results

The quantity and quality of DNA varied among the four kits. The microbiota of the stool samples showed similar diversity and compositional profiles for the 4 kits.

Conclusion

Despite differences in DNA quality and quantity, the 4 kits yielded similar results for stool samples, while all kits were not sensitive enough for low biomass samples.

SUBMITTER: Gall-David SL

PROVIDER: S-EPMC10051199 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Publications

Comparison of four DNA extraction kits efficiency for 16SrDNA microbiota profiling of diverse human samples.

Gall-David Sandrine Le SL Boudry Gaëlle G Buffet-Bataillon Sylvie S

Future science OA 20230101 1

<h4>Aim</h4>The current study investigated the performance of 4 widely used DNA extraction kits using different types of high (stool) and low biomass samples (chyme, broncho alveolar lavage and sputum).<h4>Methods</h4>Qiagen Powerfecal Pro DNA kit, Macherey Nucleospin Soil kit, Macherey Nucleospin Tissue Kit and MagnaPure LC DNA isolation kit III were evaluated in terms of DNA quantity, quality, diversity and composition profiles.<h4>Results</h4>The quantity and quality of DNA varied among the f ...[more]

PMID: 37006230

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Project description:IntroductionAdvancements in DNA extraction and sequencing technologies have been fundamental in deciphering the significance of the microbiome related to human health and pathology. Whole metagenome shotgun sequencing (WMS) is gaining popularity in use compared to its predecessor (i.e., amplicon-based approaches). However, like amplicon-based approaches, WMS is subject to bias from DNA extraction methods that can compromise the integrity of sequencing and subsequent findings. The purpose of this study was to evaluate systematic differences among four commercially available DNA extraction kits frequently used for WMS analysis of the microbiome.MethodsOral, vaginal, and rectal swabs were collected in replicates of four by a healthcare provider from five participants and randomized to one of four DNA extraction kits. Two extraction blanks and three replicate mock community samples were also extracted using each extraction kit. WMS was completed with NovaSeq 6000 for all samples. Sequencing and microbial communities were analyzed using nonmetric multidimensional scaling and compositional bias analysis.ResultsExtraction kits differentially biased the percentage of reads attributed to microbial taxa across samples and body sites. The PowerSoil Pro kit performed best in approximating expected proportions of mock communities. While HostZERO was biased against gram-negative bacteria, the kit outperformed other kits in extracting fungal DNA. In clinical samples, HostZERO yielded a smaller fraction of reads assigned to Homo sapiens across sites and had a higher fraction of reads assigned to bacterial taxa compared to other kits. However, HostZERO appears to bias representation of microbial communities and demonstrated the most dispersion by site, particularly for vaginal and rectal samples.ConclusionsSystematic differences exist among four frequently referenced DNA extraction kits when used for WMS analysis of the human microbiome. Consideration of such differences in study design and data interpretation is imperative to safeguard the integrity of microbiome research and reproducibility of results.

Project description:Next-generation sequencing (NGS) studies are becoming routinely used for the detection of novel and clinically actionable DNA variants at a pangenomic scale. Such analyses are now used in the clinical practice to enable precision medicine. Formalin-fixed paraffin-embedded (FFPE) tissues are still one of the most abundant source of cancer clinical specimen, unfortunately this method of preparation is known to degrade DNA and therefore compromise subsequent analysis. Some studies have reported that variant detection can be performed on FFPE samples sequenced with NGS techniques, but few or none have done an in-depth coverage analysis and compared the influence of different state-of-the-art FFPE DNA extraction kits on the quality of the variant calling. Here, we generated 42 human whole-exome sequencing data sets from fresh-frozen (FF) and FFPE samples. These samples include normal and tumor tissues from two different organs (liver and colon), that we extracted with three different FFPE extraction kits (QIAamp DNA FFPE Tissue kit and GeneRead DNA FFPE kit from Qiagen, Maxwell™ RSC DNA FFPE Kit from Promega). We determined the rate of concordance of called variants between matched FF and FFPE samples on all common variants (representing at least 86% of the total number of variants for SNVs). The concordance rate is very high between all matched FF / FFPE pairs, with equivalent values for the three kits we analyzed. On the other hand, when looking at the difference between the total number of variants in FF and FFPE, we find a significant variation for the three different FFPE DNA extraction kits. Coverage analysis shows that FFPE samples have less good indicators than FF samples, yet the coverage quality remains above accepted thresholds. We detect limited but statistically significant variations in coverage indicator values between the three FFPE extraction kits. Globally, the GeneRead and QIAamp kits have better variant calling and coverage indicators than the Maxwell kit on the samples used in this study, although this kit performs better on some indicators and has advantages in terms of practical usage. Taken together, our results confirm the potential of FFPE samples analysis for clinical genomic studies, but also indicate that the choice of a FFPE DNA extraction kit should be done with careful testing and analysis beforehand in order to maximize the accuracy of the results.

Dataset Information

Comparison of four DNA extraction kits efficiency for 16SrDNA microbiota profiling of diverse human samples.

Aim

Methods

Results

Conclusion

Publications

Comparison of four DNA extraction kits efficiency for 16SrDNA microbiota profiling of diverse human samples.

Similar Datasets

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