Lezione 10. Metagenomica ed environmental DNA (edna)

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1 Lezione 10 Metagenomica ed environmental DNA (edna)

2 Quali campioni e quali dati sono tipici della metagenomica? Target capture and variant calling

3 Metagenomics is the study of genetic material recovered directly from environmental samples. (Wikipedia) Metagenomics is the study of all genomes present in any given environment without the need for prior individual identification or amplification. For example, in its simplest form, a metagenomic study might be the direct sequence results of DNA extracted from a bucket of sea water. (EBI metagenomics) OTU: Operational Taxonomic Unit. The taxonomic level of sampling defined by the researcher in a study; for example, individuals, populations, species, genera, or strains.

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5 Species specific primers OR universal primer (BARCODING)

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7 Mitocondriale Nucleare Plastidiale

8 Amplificazione di un marcatore per distinguere le OTU e tag per distinguere gli individui

9 A DNA barcoding reference database, including rbcl and trnh-psba sequences from 693 plant species (104 sequenced in this study) was assembled. Pollen composition was largely influenced by floristic local biodiversity, plant phenology, and the presence of alien flowering species.

10 45 samples were isolated from slices or fillets (i.e. white muscular tissue) of sharks sold as palombo (we checked the name reported on the label) in different Italian fish markets and supermarkets. DNA marker: COI Only three (6.7%) can be unequivocally assigned to palombo and in particular to the species M. mustelus. Further, six cases out of 45 (13.3%) refer to species belonging to the genus Mustelus. Altogether, we identified 35 cases of species substitution out of 45 (77.8%) collected samples.

11 Maurizio Casiraghi Massimo Labra

12 Microbiome The microbiome comprises all of the genetic material within a microbiota (the entire collection of microorganisms in a specific niche, such as the human gut). This can also be referred to as the metagenome of the microbiota. DNA marker: 16s RNA

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14 In this work, we compared the fecal microbiota of European children (EU) and that of children from a rural African village of Burkina Faso (BF), where the diet, high in fiber content, is similar to that of early human settlements at the time of the birth of agriculture. By using high-throughput 16S rdna sequencing and biochemical analyses, we found significant differences in gut microbiota between the two groups.

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16 BF children showed a significant enrichment in Bacteroidetes and depletion in Firmicutes (P < 0.001), with a unique abundance of bacteria from the genus Prevotella and Xylanibacter, known to contain a set of bacterial genes for cellulose and xylan hydrolysis, completely lacking in the EU children We hypothesize that gut microbiota coevolved with the polysacchariderich diet of BF individuals, allowing them to maximize energy intake from fibers while also protecting them from inflammations and noninfectious colonic diseases. This study investigates and compares human intestinal microbiota from children characterized by a modern western diet and a rural diet, indicating the importance of preserving this treasure of microbial diversity from ancient rural communities worldwide.

17 PubMed search: microbiome AND human disease 5992 articoli

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19 Environmental DNA (edna) trace DNA in samples such as water, soil, or faeces. edna is a mixture of potentially degraded DNA from many different organisms. Bohmann et al TEE

20 Bohmann et al TEE

21 Bohmann et al TEE

22 sanguisuga Bohmann et al TEE

23 idna > edna (idna= invertebrate) Bohmann et al TEE

24 Bohmann et al TEE

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30 Bioinformatica: i file fastq contengono sequenze (reads) di più organismi per lo stesso GAGTGGACTCCTGCGGGAGGCAGCAGTGGGGAATCTTCGGCAATGGGCGCAAGCCTGACCGAGCCATGCCGCGTGAGTGAAGAAGGCCTTAGGGTCGT AAAGCTCTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGGTAGCGTT GTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGACTATTAAGTCAGGGGTGAAATCCCGGGGCTCAACCCCGGAACTGCCTTGTAAACTCCCAGT CTAGAG + CCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGE GGGGGGGGGGGGGGGGGGFFGGFGGGGGGGGGGFGGGFGFFGGGFFGGGGGGGGGGGFGGG@FGGGGGDGGFGGGG@FEGGGGFGGGGGG*?858>*A EEGGGGGGGGGGGFGG5CCEGDFGC7DG:*:EEGGGDG5CFFGGFF467?FB>D:*@>D?F2>)<777?F:F8>>5)- GAGTGGACTCCTACGGGAGGCAGCAGTGGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGTGTGTGATGAAGGCCTTAGGGTCGT AAAGCACTTTCGCCAGGGATGATAATGACAGTACCTGGTAAAGAAACCCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGTTAGCGTT GTTCGGAATTACTGGGCGTAAAGCGTACGTAGGCGGATTAGAAAGTATAGGGTGAAATCCCTGGGCTCAACCCAGGAACTGCCTTGTAAACTCCTAGT CTTGAG + CCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGFGGGGGGGFGGFGGE GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGEGGGGGGGGFGGGGGGGEGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG5EEGGGGGGGGGGG GGGGGGGGGGGGFGGGGGEGDCGG5EGGGGGG:09DGGCCFB9>@FFFGFGF@<>6@GG6F>FFD5)C6C5F:<>>3:56>BFFBFFFA>BA<>DFF4 GAGTGGACTCCTACGGGAGGCAGCAGTGAGGAATATTGCGCAATGGGGGAAACCCTGACGCAGCGACGCCGCGTGGATGATGAAGGCCTTCGGGTTGT AAAATCCTGTCAGATGGGAAGAAATGCTTTATGCCTAATACGTGTAGAGTTTGACGGTACCATCAAAGGAAGCACCGGCTAACTCCGTGCCAGCAGCC GCGGTAATACGGAGGGTGCGAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCGCGTAGGCGGCTATTTAAGTCAGATGTGAAAGTCCACGGCCCAACC GTGGAA + CCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGFGGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGFFFGGGGGGGGGGGGFGCGGGGFGFGGGGGGGGGFGGGGGGFEGGGGGGGG GGGDE8CFGGGGEEGEEEGGDGGGEGGGGGGGFGGGGGGFGFGGFEFFGF37;DD<DDGFGF@>9?FFFGF?F>6**.8@<577<*=A??9;(, ;6) Sono ampliconi della stessa lunghezza

31 What are the microorganism capable of doing as a community? Alpha: the mean species diversity in sites or habitats at a local scale Beta: ratio between regional and local species diversity. Comparing different environments

32 Uso di uno specifico marcatore: problemi -bisogna disegnare primers che vadano bene per molte OTU -bisogna trovare il giusto marcatore (che sia cioè in grado di distinguere tra diverse OTU) -bisogna amplificare specificamente -questo si può superare con il capturing (ricordate le lezioni pratiche? Richiede meno specificità di una PCR) -bisogna avere un database di riferimento che contenga quel marcatore per diversi organismi

33 Con i nuovi metodi di sequenziamento e l aumento di informazioni nelle banche dati si può passare ad uno shotgun sequencing (simile ad un sequenziamento genomico, ma di tutto ciò che è stato raccolto nel campione) (vedere il progetto rg/) D (A) Sampling from habitat (B) filtering particles, typically by size (C) DNA extraction and lysis (D) sequencing (E) sequence assembly. E

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