Dataset Information

Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

ABSTRACT:

Background

Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples.

Results

In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories.

Conclusions

The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.

SUBMITTER: Tourlousse DM

PROVIDER: S-EPMC8082873 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Publications

Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

Tourlousse Dieter M DM Narita Koji K Miura Takamasa T Sakamoto Mitsuo M Ohashi Akiko A Shiina Keita K Matsuda Masami M Miura Daisuke D Shimamura Mamiko M Ohyama Yoshifumi Y Yamazoe Atsushi A Uchino Yoshihito Y Kameyama Keishi K Arioka Shingo S Kataoka Jiro J Hisada Takayoshi T Fujii Kazuyuki K Takahashi Shunsuke S Kuroiwa Miho M Rokushima Masatomo M Nishiyama Mitsue M Tanaka Yoshiki Y Fuchikami Takuya T Aoki Hitomi H Kira Satoshi S Koyanagi Ryo R Naito Takeshi T Nishiwaki Morie M Kumagai Hirotaka H Konda Mikiko M Kasahara Ken K Ohkuma Moriya M Kawasaki Hiroko H Sekiguchi Yuji Y Terauchi Jun J

Microbiome 20210429 1

<h4>Background</h4>Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples.<h4>Results</h4>In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction ...[more]

PMID: 33910647

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Project description:Microbiome studies are becoming larger in size to detect the potentially small effect that environmental factors have on our gut microbiomes, or that the microbiome has on our health. Therefore, fast and reproducible DNA isolation methods are needed to handle thousands of fecal samples. We used the Chemagic 360 chemistry and Magnetic Separation Module I (MSMI) instrument to compare two sample preservatives and four different pre-treatment protocols to find an optimal method for DNA isolation from thousands of fecal samples. The pre-treatments included bead beating, sample handling in tube and plate format, and proteinase K incubation. The optimal method offers a sufficient yield of high-quality DNA without contamination. Three human fecal samples (adult, senior, and infant) with technical replicates were extracted. The extraction included negative controls (OMNIgeneGUT, DNA/RNA shield fluid, and Chemagic Lysis Buffer 1) to detect cross-contamination and ZymoBIOMICS Gut Microbiome Standard as a positive control to mimic the human gut microbiome and assess sensitivity of the extraction method. All samples were extracted using Chemagic DNA Stool 200 H96 kit (PerkinElmer, Finland). The samples were collected in two preservatives, OMNIgeneGUT and DNA/RNA shield fluid. DNA quantity was measured using Qubit-fluorometer, DNA purity and quality using gel electrophoresis, and taxonomic signatures with 16S rRNA gene-based sequencing with V3V4 and V4 regions. Bead beating increased bacterial diversity. The largest increase was detected in gram-positive genera Blautia, Bifidobacterium, and Ruminococcus. Preservatives showed minor differences in bacterial abundances. The profiles between the V3V4 and V4 regions differed considerably with lower diversity samples. Negative controls showed signs from genera abundant in fecal samples. Technical replicates of the Gut Standard and stool samples showed low variation. The selected isolation protocol included recommended steps from manufacturer as well as bead beating. Bead beating was found to be necessary to detect hard-to-lyse bacteria. The protocol was reproducible in terms of DNA yield among different stool replicates and the ZymoBIOMICS Gut Microbiome Standard. The MSM1 instrument and pre-treatment in a 96-format offered the possibility of automation and handling of large sample collections. Both preservatives were feasible in terms of sample handling and had low variation in taxonomic signatures. The 16S rRNA target region had a high impact on the composition of the bacterial profile.ImportanceNext-generation sequencing (NGS) is a widely used method for determining the composition of the gut microbiota. Due to the differences in the gut microbiota composition between individuals, microbiome studies have expanded into large population studies to maximize detection of small effects on microbe-host interactions. Thus, the demand for a rapid and reliable microbial profiling is continuously increasing, making the optimization of high-throughput 96-format DNA extraction integral for NGS-based downstream applications. However, experimental protocols are prone to bias and errors from sample collection and storage, to DNA extraction, primer selection and sequencing, and bioinformatics analyses. Methodological bias can contribute to differences in microbiome profiles, causing variability across studies and laboratories using different protocols. To improve consistency and confidence of the measurements, the standardization of microbiome analysis methods has been recognized in many fields.

Dataset Information

Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

Background

Results

Conclusions

Publications

Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

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