Lezione 8

Metagenomica ed environmental DNA (eDNA)

Quali campioni e quali dati sono tipici della metagenomica?

Target capture and variant calling

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 manydifferent organisms found in an environmental sample

• Microbiome: all the microbes in a community

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.

https://www.ebi.ac.uk/metagenomics/

Shot-gun(sequenzio tutto!)

Amplicon-based

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 http://extrememicrobiome.org/)

(A) Sampling from habitat(B) filtering particles, typically by size(C) DNA extraction and lysis(D) sequencing (E) sequence assembly.

http://blog.booleanbiotech.com/nanopore_2016.html

D

E

Why? Think…see slide 47

http://www.boldsystems.org

Mitocondriale

Nucleare

Plastidiale

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

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

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.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0109363

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.

Mustelus mustelus

Squalus acanthias

Isurus oxyrinchus

http://www.zooplantlab.btbs.unimib.it/

Maurizio Casiraghi

Massimo Labra

Environmental DNA

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. 2014 TEE

Bohmann et al. 2014 TEE

Bohmann et al. 2014 TEE

sanguisuga

Bohmann et al. 2014 TEE

iDNA > eDNA

(iDNA= invertebrate)

Bohmann et al. 2014 TEE

Bohmann et al. 2014 TEE

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

http://microbiomaitaliano.it/

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.

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 ofbacterial genes for cellulose and xylan hydrolysis, completely lackingin the EU children

We hypothesize that gut microbiota coevolved with the polysaccharide-rich 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.

PubMed search:

microbiome AND human disease

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