Systematic Cross-Biospecimen Evaluation of DNA Extraction Kits for Long- and Short-Read Multi-Metagenomic Sequencing Studies

Rehner, Jacqueline; Schmartz, Georges Pierre; Groeger, Laura; Dastbaz, Jan; Ludwig, Nicole; Hannig, Matthias; Rupf, Stefan; Seitz, Berthold; Flockerzi, Elias; Berger, Tim; Reichert, Matthias; Krawczyk, Marcin; Meese, Eckart; Herr, Christian; Bals, Robert; Becker, Sören L.; Keller, Andreas; Müller, Rolf

doi:10.1016/j.gpb.2022.05.006

Cited by 5 publications

(1 citation statement)

References 38 publications

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“…We extracted whole-genome DNA from all faecal samples using the ZymoBIOMICS DNA Miniprep Kit [25]. DNA was isolated and purified according to the manufacturer's protocol.…”

Section: Dna Extractionmentioning

confidence: 99%

The Effect of a Planetary Health Diet on the Human Gut Microbiome: A Descriptive Analysis

et al. 2023

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In 2019, researchers from the EAT-Lancet Commission developed the ‘Planetary Health (PH) diet’. Specifically, they provided recommendations pertaining to healthy diets derived from sustainable food systems. Thus far, it has not been analysed how such a diet affects the human intestinal microbiome, which is important for health and disease development. Here, we present longitudinal genome-wide metagenomic sequencing and mass spectrometry data on the gut microbiome of healthy volunteers adhering to the PH diet, as opposed to vegetarian or vegan (VV) and omnivorous (OV) diets. We obtained basic epidemiological information from 41 healthy volunteers and collected stool samples at inclusion and after 2, 4, and 12 weeks. Individuals opting to follow the PH diet received detailed instructions and recipes, whereas individuals in the control groups followed their habitual dietary pattern. Whole-genome DNA was extracted from stool specimens and subjected to shotgun metagenomic sequencing (~3 GB per patient). Conventional bacterial stool cultures were performed in parallel and bacterial species were identified with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. We analysed samples from 16 PH, 16 OV, and 9 VV diet patterns. The α-diversity remained relatively stable for all dietary groups. In the PH group, we observed a constant increase from 3.79% at inclusion to 4.9% after 12 weeks in relative abundance of Bifidobacterium adolescentis. Differential PH abundance analysis highlighted a non-significant increase in possible probiotics such as Paraprevotella xylaniphila and Bacteroides clarus. The highest abundance of these bacteria was observed in the VV group. Dietary modifications are associated with rapid alterations to the human gut microbiome, and the PH diet led to a slight increase in probiotic-associated bacteria at ≥4 weeks. Additional research is required to confirm these findings.

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“…We extracted whole-genome DNA from all faecal samples using the ZymoBIOMICS DNA Miniprep Kit [25]. DNA was isolated and purified according to the manufacturer's protocol.…”

Section: Dna Extractionmentioning

confidence: 99%

The Effect of a Planetary Health Diet on the Human Gut Microbiome: A Descriptive Analysis

et al. 2023

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Mibianto: ultra-efficient online microbiome analysis through k-mer based metagenomics

Hirsch,

Molano,

Engel

et al. 2024

Nucleic Acids Research

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Quantifying microbiome species and composition from metagenomic assays is often challenging due to its time-consuming nature and computational complexity. In Bioinformatics, k-mer-based approaches were long established to expedite the analysis of large sequencing data and are now widely used to annotate metagenomic data. We make use of k-mer counting techniques for efficient and accurate compositional analysis of microbiota from whole metagenome sequencing. Mibianto solves this problem by operating directly on read files, without manual preprocessing or complete data exchange. It handles diverse sequencing platforms, including short single-end, paired-end, and long read technologies. Our sketch-based workflow significantly reduces the data volume transferred from the user to the server (up to 99.59% size reduction) to subsequently perform taxonomic profiling with enhanced efficiency and privacy. Mibianto offers functionality beyond k-mer quantification; it supports advanced community composition estimation, including diversity, ordination, and differential abundance analysis. Our tool aids in the standardization of computational workflows, thus supporting reproducibility of scientific sequencing studies. It is adaptable to small- and large-scale experimental designs and offers a user-friendly interface, thus making it an invaluable tool for both clinical and research-oriented metagenomic studies. Mibianto is freely available without the need for a login at: https://www.ccb.uni-saarland.de/mibianto.

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Comparison of 6 DNA extraction methods for isolation of high yield of high molecular weight DNA suitable for shotgun metagenomics Nanopore sequencing to detect bacteria

Gand¹,

Bloemen²,

Vanneste³

et al. 2023

BMC Genomics

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Background Oxford Nanopore Technologies (ONT) offers an accessible platform for long-read sequencing, which improves the reconstruction of genomes and helps to resolve complex genomic contexts, especially in the case of metagenome analysis. To take the best advantage of long-read sequencing, DNA extraction methods must be able to isolate pure high molecular weight (HMW) DNA from complex metagenomics samples, without introducing any bias. New methods released on the market, and protocols developed at the research level, were specifically designed for this application and need to be assessed. Results In this study, with different bacterial cocktail mixes, analyzed as pure or spiked in a synthetic fecal matrix, we evaluated the performances of 6 DNA extraction methods using various cells lysis and purification techniques, from quick and easy, to more time-consuming and gentle protocols, including a portable method for on-site application. In addition to the comparison of the quality, quantity and purity of the extracted DNA, the performance obtained when doing Nanopore sequencing on a MinION flow cell was also tested. From the obtained results, the Quick-DNA HMW MagBead Kit (Zymo Research) was selected as producing the best yield of pure HMW DNA. Furthermore, this kit allowed an accurate detection, by Nanopore sequencing, of almost all the bacterial species present in a complex mock community. Conclusion Amongst the 6 tested methods, the Quick-DNA HMW MagBead Kit (Zymo Research) was considered as the most suitable for Nanopore sequencing and would be recommended for bacterial metagenomics studies using this technology.

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Systematic Cross-Biospecimen Evaluation of DNA Extraction Kits for Long- and Short-Read Multi-Metagenomic Sequencing Studies

Cited by 5 publications

References 38 publications

The Effect of a Planetary Health Diet on the Human Gut Microbiome: A Descriptive Analysis

The Effect of a Planetary Health Diet on the Human Gut Microbiome: A Descriptive Analysis

Mibianto: ultra-efficient online microbiome analysis through k-mer based metagenomics

Comparison of 6 DNA extraction methods for isolation of high yield of high molecular weight DNA suitable for shotgun metagenomics Nanopore sequencing to detect bacteria

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