Metagenomics: Methods and Protocols

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The Adonis function in the Vegan library v2. This implements a permutational multivariate analysis of variance PERMANOVA , which allows for identifying associations between distance matrices and meta data variables assigning significance through a permutation-based test [ 83 ]. Revised estimates for the number of human and bacteria cells in the body.

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New Protocol: Metagenomic Profiling with SMRT Sequencing

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The first cultured species of the human gastrointestinal microbiota. Metagenomic analyses of an uncultured viral community from human feces. J Bacteriol. Viral diversity and dynamics in an infant gut. Res Microbiol. Enterotypes of the human gut microbiome. A human gut microbial gene catalogue established by metagenomic sequencing. Diversity and Abundance of single-stranded DNA viruses in human feces. Appl Environ Microbiol. Characterization of virus-like particles associated with the human faecal and caecal microbiota.

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Metagenomics: Methods and Protocols - Methods in Molecular Biology Vol 668

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Methods and Protocols

Anderson MJ. A new method for non-parametric multivariate analysis of variance. Austral Ecol. Download references. All authors read and approved the final manuscript. Correspondence to Colin Hill. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Table S1. Comparison of methods used in published metagenomic studies of viral communities associated with various sites in the human body.

XLSX 14 kb. Table S2. Description of faecal samples used in the study and summary of nucleic acid extraction and sequencing results. XLSX kb. Figure S1. Percentage of reads aligned to conserved segment of bacterial cpn60 gene and rRNA genes operon in virome samples from four separate experiments within this study compared to a selection of 18 total metagenomic samples sequenced as part of the Human Microbiome Project. PDF 29 kb. Figure S2.

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Effect of storage on the composition of faecal phageomes and bacteriomes continued from Fig. E and F, Barplots of relative abundance of bacterial taxonomic groups at the genus level and viruses at the individual contig level. PDF kb. Table S3. DESeq2 test results for differential abundance of putative phage contigs in six faecal samples processed three different operators O1, O2 and O3. Figure S3. Effect of repeated freeze-thaw cycles on the composition of faecal phageomes and bacteriomes continued from Fig.

E, Barplot of relative abundance of bacterial taxonomic groups at the genus level. Figure S4. Operator-dependent variations in faecal phageome and bacteriome profiling results continued from Fig. NMDS stress values were 0. E and F, Barplots of relative abundance of bacterial taxonomic groups at the genus level and viruses at the individual contigs level. Reprints and Permissions. Search all BMC articles Search. Shkoporov 1 , Feargal J. Ryan 1 , Lorraine A. Draper 1 , Amanda Forde 1 , Stephen R. Stockdale 2 , Karen M. Daly 1 , Siobhan A.

McDonnell 1 , James A. Nolan 1 , Thomas D.

Tissue-Based Universal Virus Detection (TUViD-VM) Protocol for Viral Metagenomics.

Abstract Background Recent studies have demonstrated that the human gut is populated by complex, highly individual and stable communities of viruses, the majority of which are bacteriophages. Results Faecal phageome profiles exhibit an overall greater individual specificity when compared to bacteriome profiles. Background The last two decades have seen tremendous progress in understanding the structure, dynamics and physiological significance of diverse microbial communities populating the human gastro-intestinal tract GIT , collectively known as the gut microbiome.

Results Design of the experiments To investigate the relative impact of faecal sample storage conditions, repeated freeze-thaw cycles and operator-dependent bias on the outcome of gut phageome profiles, three separate and independent experiments were conducted on subsamples of faecal samples collected from 10 healthy adult volunteers and 3 adult IBD patients. Full size image.

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Discussion Since the first attempts of metagenomic analysis of viral communities in the gut by Breitbart and colleagues more than a decade ago [ 12 ], a considerable amount of sequence data has accumulated in public repositories. Conclusions The overall effect observed in the present study was a greater relative individual specificity of phageome profiles obtained using shotgun metagenomic sequencing of faecal VLP fractions compared to bacteriome profiles obtained using amplicon sequencing of 16S rRNA gene fragments.

Storage experiment Fresh faecal samples were collected from four healthy adults. Operator reproducibility experiment Frozen faecal samples collected from 3 healthy adults and 3 IBD patients were processed twice on two separate days by each of the three operators. Faecal VLP nucleic acid extraction Aliquots of 0. Analysis of phageome shotgun sequencing data The quality of the raw reads was visualised with FastQC v0.

Statistical analysis Alpha diversity and beta diversity were generated using PhyloSeq v1. References 1. Article PubMed Google Scholar 7. Google Scholar Article Google Scholar Article PubMed Google Scholar Google Scholar Download references. Cork, Ireland Stephen R. Consent for publication Not applicable. Competing interests The authors declared that they have no competing interests. Additional files Additional file 1: Table S1.

Additional file 2: Table S2. Additional file 3: Figure S1. Additional file 4: Figure S2.


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