Lezione 8. Metagenomica ed environmental DNA (edna)

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1 Lezione 8 Metagenomica ed environmental DNA (edna)

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

3 Alcune definizioni Metagenomics: is the study of all genomes present in any given environment without the need for prior individual identification or amplification DNA barcoding: DNA barcoding is a taxonomic method, that uses one or more standardized short genetic markers in an organism s DNA to identify it as belonging to a particular species. Through this method unknown DNA samples are identified to registered species based on comparison to a reference dataset Metabarcoding: biodiversity assessment using DNA barcodes Environmental DNA (edna): complex mixture of genomic DNA from many different organisms found in an environmental sample Microbiome: all the microbes in a community

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5 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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7 Shot-gun (sequenzio tutto!) Amplicon-based

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9 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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11 Why? Think see slide 47

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14 Mitocondriale Nucleare Plastidiale

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

16 DNA metabarcoding: 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 bisogna avere un database di riferimento che contenga quel marcatore per diversi organismi la PCR fa errori, più di un sequenziamento Se il DNA è degradato è difficile amplificare marcatori lunghi > 200 bp Se si fa una PCR si perde l informazione dell abbondanza relativa

17 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.

18 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. Mustelus mustelus 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. Squalus acanthias Isurus oxyrinchus

19 Maurizio Casiraghi Massimo Labra

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22 Environmental DNA

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24 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

25 Bohmann et al TEE

26 Bohmann et al TEE

27 sanguisuga Bohmann et al TEE

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

29 Bohmann et al TEE

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35 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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38 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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40 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.

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43 PubMed search: microbiome AND human disease 5992 articoli (2016) articoli (2018)

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