Microbiota

# .font170[MICROBIOMA E MICROBIOTA]

<br><br><br><br>
.marco[
Marco Chiapello
<br>
2020-11-17
]

.marco[
.center[.font90[[[<svg style="height:0.8em;top:.04em;position:relative;fill:steelblue;" viewBox="0 0 448 512"><path d="M94.12 315.1c0 25.9-21.16 47.06-47.06 47.06S0 341 0 315.1c0-25.9 21.16-47.06 47.06-47.06h47.06v47.06zm23.72 0c0-25.9 21.16-47.06 47.06-47.06s47.06 21.16 47.06 47.06v117.84c0 25.9-21.16 47.06-47.06 47.06s-47.06-21.16-47.06-47.06V315.1zm47.06-188.98c-25.9 0-47.06-21.16-47.06-47.06S139 32 164.9 32s47.06 21.16 47.06 47.06v47.06H164.9zm0 23.72c25.9 0 47.06 21.16 47.06 47.06s-21.16 47.06-47.06 47.06H47.06C21.16 243.96 0 222.8 0 196.9s21.16-47.06 47.06-47.06H164.9zm188.98 47.06c0-25.9 21.16-47.06 47.06-47.06 25.9 0 47.06 21.16 47.06 47.06s-21.16 47.06-47.06 47.06h-47.06V196.9zm-23.72 0c0 25.9-21.16 47.06-47.06 47.06-25.9 0-47.06-21.16-47.06-47.06V79.06c0-25.9 21.16-47.06 47.06-47.06 25.9 0 47.06 21.16 47.06 47.06V196.9zM283.1 385.88c25.9 0 47.06 21.16 47.06 47.06 0 25.9-21.16 47.06-47.06 47.06-25.9 0-47.06-21.16-47.06-47.06v-47.06h47.06zm0-23.72c-25.9 0-47.06-21.16-47.06-47.06 0-25.9 21.16-47.06 47.06-47.06h117.84c25.9 0 47.06 21.16 47.06 47.06 0 25.9-21.16 47.06-47.06 47.06H283.1z"/></svg>](https://join.slack.com/t/ipma-2020/shared_invite/zt-j9o8ig7k-LN1~U3d6QJztquYbC6iW7w) Slack (https://bit.ly/3eRDBRL) - <svg style="height:0.8em;top:.04em;position:relative;fill:steelblue;" viewBox="0 0 512 512"><path d="M502.3 190.8c3.9-3.1 9.7-.2 9.7 4.7V400c0 26.5-21.5 48-48 48H48c-26.5 0-48-21.5-48-48V195.6c0-5 5.7-7.8 9.7-4.7 22.4 17.4 52.1 39.5 154.1 113.6 21.1 15.4 56.7 47.8 92.2 47.6 35.7.3 72-32.8 92.3-47.6 102-74.1 131.6-96.3 154-113.7zM256 320c23.2.4 56.6-29.2 73.4-41.4 132.7-96.3 142.8-104.7 173.4-128.7 5.8-4.5 9.2-11.5 9.2-18.9v-19c0-26.5-21.5-48-48-48H48C21.5 64 0 85.5 0 112v19c0 7.4 3.4 14.3 9.2 18.9 30.6 23.9 40.7 32.4 173.4 128.7 16.8 12.2 50.2 41.8 73.4 41.4z"/></svg>](mailto:marco.chiapello@unito.it) marco.chiapello@unito.it]
]
]

---
layout: true

# Last lesson check

---

---

# .black[Il microbiota vegetale varia?]

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      <td class="gt_row gt_left">Si, ma solo tra la composizione del microbiota presente nella rizosfera e e quello dell'endosfera</td>
      <td class="gt_row gt_center">1 <svg style="height:0.8em;top:.04em;position:relative;fill:red;" viewBox="0 0 512 512"><path d="M256 8C119 8 8 119 8 256s111 248 248 248 248-111 248-248S393 8 256 8zm121.6 313.1c4.7 4.7 4.7 12.3 0 17L338 377.6c-4.7 4.7-12.3 4.7-17 0L256 312l-65.1 65.6c-4.7 4.7-12.3 4.7-17 0L134.4 338c-4.7-4.7-4.7-12.3 0-17l65.6-65-65.6-65.1c-4.7-4.7-4.7-12.3 0-17l39.6-39.6c4.7-4.7 12.3-4.7 17 0l65 65.7 65.1-65.6c4.7-4.7 12.3-4.7 17 0l39.6 39.6c4.7 4.7 4.7 12.3 0 17L312 256l65.6 65.1z"/></svg></td>
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      <td class="gt_row gt_left">Si, varia nel tempo. In diversi momenti della vita della pianta la composizione del microbiota muta</td>
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]

---

# .black[Qual è la forza selettiva maggiore che da forma al microbiota associato alle piante?]

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<div id="kuqeuokwje" style="overflow-x:auto;overflow-y:auto;width:auto;height:auto;"><table class="gt_table">
  
  <thead class="gt_col_headings">
    <tr>
      <th class="gt_col_heading gt_columns_bottom_border gt_left" rowspan="1" colspan="1">Opzioni</th>
      <th class="gt_col_heading gt_columns_bottom_border gt_center" rowspan="1" colspan="1">Risposte</th>
    </tr>
  </thead>
  <tbody class="gt_table_body">
    <tr>
      <td class="gt_row gt_left">Il microbiota</td>
      <td class="gt_row gt_center">4 <svg style="height:0.8em;top:.04em;position:relative;fill:red;" viewBox="0 0 512 512"><path d="M256 8C119 8 8 119 8 256s111 248 248 248 248-111 248-248S393 8 256 8zm121.6 313.1c4.7 4.7 4.7 12.3 0 17L338 377.6c-4.7 4.7-12.3 4.7-17 0L256 312l-65.1 65.6c-4.7 4.7-12.3 4.7-17 0L134.4 338c-4.7-4.7-4.7-12.3 0-17l65.6-65-65.6-65.1c-4.7-4.7-4.7-12.3 0-17l39.6-39.6c4.7-4.7 12.3-4.7 17 0l65 65.7 65.1-65.6c4.7-4.7 12.3-4.7 17 0l39.6 39.6c4.7 4.7 4.7 12.3 0 17L312 256l65.6 65.1z"/></svg></td>
    </tr>
    <tr>
      <td class="gt_row gt_left">L'evoluzione</td>
      <td class="gt_row gt_center">7 <svg style="height:0.8em;top:.04em;position:relative;fill:red;" viewBox="0 0 512 512"><path d="M256 8C119 8 8 119 8 256s111 248 248 248 248-111 248-248S393 8 256 8zm121.6 313.1c4.7 4.7 4.7 12.3 0 17L338 377.6c-4.7 4.7-12.3 4.7-17 0L256 312l-65.1 65.6c-4.7 4.7-12.3 4.7-17 0L134.4 338c-4.7-4.7-4.7-12.3 0-17l65.6-65-65.6-65.1c-4.7-4.7-4.7-12.3 0-17l39.6-39.6c4.7-4.7 12.3-4.7 17 0l65 65.7 65.1-65.6c4.7-4.7 12.3-4.7 17 0l39.6 39.6c4.7 4.7 4.7 12.3 0 17L312 256l65.6 65.1z"/></svg></td>
    </tr>
    <tr>
      <td class="gt_row gt_left">La pianta</td>
      <td class="gt_row gt_center">8 <svg style="height:0.8em;top:.04em;position:relative;fill:green;" viewBox="0 0 512 512"><path d="M504 256c0 136.967-111.033 248-248 248S8 392.967 8 256 119.033 8 256 8s248 111.033 248 248zM227.314 387.314l184-184c6.248-6.248 6.248-16.379 0-22.627l-22.627-22.627c-6.248-6.249-16.379-6.249-22.628 0L216 308.118l-70.059-70.059c-6.248-6.248-16.379-6.248-22.628 0l-22.627 22.627c-6.248 6.248-6.248 16.379 0 22.627l104 104c6.249 6.249 16.379 6.249 22.628.001z"/></svg></td>
    </tr>
  </tbody>
  
  
</table></div>
]

---

# .black[Per olobiote si intende...]

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<div id="feopepohgp" style="overflow-x:auto;overflow-y:auto;width:auto;height:auto;"><table class="gt_table">
  
  <thead class="gt_col_headings">
    <tr>
      <th class="gt_col_heading gt_columns_bottom_border gt_left" rowspan="1" colspan="1">Opzioni</th>
      <th class="gt_col_heading gt_columns_bottom_border gt_center" rowspan="1" colspan="1">Risposte</th>
    </tr>
  </thead>
  <tbody class="gt_table_body">
    <tr>
      <td class="gt_row gt_left">L'insieme del genoma dell'organismo ospite e del genoma dei microrganismi che lo abitano</td>
      <td class="gt_row gt_center">7 <svg style="height:0.8em;top:.04em;position:relative;fill:red;" viewBox="0 0 512 512"><path d="M256 8C119 8 8 119 8 256s111 248 248 248 248-111 248-248S393 8 256 8zm121.6 313.1c4.7 4.7 4.7 12.3 0 17L338 377.6c-4.7 4.7-12.3 4.7-17 0L256 312l-65.1 65.6c-4.7 4.7-12.3 4.7-17 0L134.4 338c-4.7-4.7-4.7-12.3 0-17l65.6-65-65.6-65.1c-4.7-4.7-4.7-12.3 0-17l39.6-39.6c4.7-4.7 12.3-4.7 17 0l65 65.7 65.1-65.6c4.7-4.7 12.3-4.7 17 0l39.6 39.6c4.7 4.7 4.7 12.3 0 17L312 256l65.6 65.1z"/></svg></td>
    </tr>
    <tr>
      <td class="gt_row gt_left">Un organismo vivente alla luce della convivenza con il proprio microbiota</td>
      <td class="gt_row gt_center">6 <svg style="height:0.8em;top:.04em;position:relative;fill:green;" viewBox="0 0 512 512"><path d="M504 256c0 136.967-111.033 248-248 248S8 392.967 8 256 119.033 8 256 8s248 111.033 248 248zM227.314 387.314l184-184c6.248-6.248 6.248-16.379 0-22.627l-22.627-22.627c-6.248-6.249-16.379-6.249-22.628 0L216 308.118l-70.059-70.059c-6.248-6.248-16.379-6.248-22.628 0l-22.627 22.627c-6.248 6.248-6.248 16.379 0 22.627l104 104c6.249 6.249 16.379 6.249 22.628.001z"/></svg></td>
    </tr>
    <tr>
      <td class="gt_row gt_left">membri del microbiota che sono presenti in tutte, o quasi, le comunità associate ad una determinato ospite</td>
      <td class="gt_row gt_center">6 <svg style="height:0.8em;top:.04em;position:relative;fill:red;" viewBox="0 0 512 512"><path d="M256 8C119 8 8 119 8 256s111 248 248 248 248-111 248-248S393 8 256 8zm121.6 313.1c4.7 4.7 4.7 12.3 0 17L338 377.6c-4.7 4.7-12.3 4.7-17 0L256 312l-65.1 65.6c-4.7 4.7-12.3 4.7-17 0L134.4 338c-4.7-4.7-4.7-12.3 0-17l65.6-65-65.6-65.1c-4.7-4.7-4.7-12.3 0-17l39.6-39.6c4.7-4.7 12.3-4.7 17 0l65 65.7 65.1-65.6c4.7-4.7 12.3-4.7 17 0l39.6 39.6c4.7 4.7 4.7 12.3 0 17L312 256l65.6 65.1z"/></svg></td>
    </tr>
  </tbody>
  
  
</table></div>
]

---
layout: true

# Last lesson recap

---

1. Plant compartment is a major selective force that shapes the composition of plant-associated microbiota

1. Plants and their associated microorganisms form a holobiont

1. Core and Hub microbiota

]
]
]

---

1. .opacity10[ Plant compartment is a major selective force that shapes the composition of plant-associated microbiota]

1. .opacity10[ Plants and their associated microorganisms form a holobiont]

1. .opacity10[ Core and Hub microbiota]

]
]
]

.m0p[
<img src="images/PlantMicrobiomeComposition.png" width="230px" height="230px" style="display: block; margin: auto;" />

<img src="images/plantMicrobiotaTemporal.png" width="300px" height="270px" style="display: block; margin: auto;" />
]
]

---

1. Plant compartment is a major selective force that shapes the composition of plant-associated microbiota

1. .opacity10[ Plants and their associated microorganisms form a holobiont]

1. .opacity10[ Core and Hub microbiota]

]
]
]

<img src="images/fungalDiv.png" width="350px" style="display: block; margin: auto;" />
]

---

1. .opacity10[ Plant compartment is a major selective force that shapes the composition of plant-associated microbiota]

1. Plants and their associated microorganisms form a holobiont

1. .opacity10[ Core and Hub microbiota]

]
]
]

.pull-right[
<br>
<img src="images/holobiont1.png" width="800px" style="display: block; margin: auto;" />

]

---

1. .opacity10[ Plant compartment is a major selective force that shapes the composition of plant-associated microbiota]

1. .opacity10[Plants and their associated microorganisms form a holobiont]

1. Core and Hub microbiota

]
]
]

]

---
layout: false
class: clear

- .font140[How do you study the microbiota composition?]

- .font140[What can the microbiota potentially do?]

- .font140[What are they doing?]
]

???

.h201[
- Questa lezione prevede dei capitoli che riguardano la **pratica** dello studio del microbiota

- Nel primo capitolo vedremo come si rispone alla domanda: "Chi è presente?"

- Nel secondo capitolo risponderemo alla domanda: "Quali funzioni possono avere i microorganismi che compongono il microbiota?"

- Ed infine risponderemo alla domanda, forse, più importante: "Che cosa STANNO facendo i microorganismi presenti nel microbiota"
]

---
layout: true

# How do you study the microbiota composition?

---
class: inverse, middle, center

----

???

- Rispondiamo quindi alla domanda: "Chi è presente?"
]

---

.h20[
<img src="images/microbiotaCompositionTecniques.png" width="520px" style="display: block; margin: auto;" />

???

.h201[
- Le 2 metodologie di studio del microbiota si basano su 2 principi diametralmente opposti:
  - Da una parta vedremo un approccio indipendete dalla coltura in vitro ma basato sul materiale genetico     
  - Dall'altra un approccio esclusivamente incentrato sulla coltura in vitro
]

---

.pull-left[
<img src="images/microbiotaCompositionTecniques.png" width="520px" style="display: block; margin: auto;" />
]

- <svg style="height:0.8em;top:.04em;position:relative;fill:green;" viewBox="0 0 512 512"><path d="M256 8C119 8 8 119 8 256s111 248 248 248 248-111 248-248S393 8 256 8zm144 276c0 6.6-5.4 12-12 12h-92v92c0 6.6-5.4 12-12 12h-56c-6.6 0-12-5.4-12-12v-92h-92c-6.6 0-12-5.4-12-12v-56c0-6.6 5.4-12 12-12h92v-92c0-6.6 5.4-12 12-12h56c6.6 0 12 5.4 12 12v92h92c6.6 0 12 5.4 12 12v56z"/></svg> This approach isolates individual microbes

- <svg style="height:0.8em;top:.04em;position:relative;fill:red;" viewBox="0 0 512 512"><path d="M256 8C119 8 8 119 8 256s111 248 248 248 248-111 248-248S393 8 256 8zm121.6 313.1c4.7 4.7 4.7 12.3 0 17L338 377.6c-4.7 4.7-12.3 4.7-17 0L256 312l-65.1 65.6c-4.7 4.7-12.3 4.7-17 0L134.4 338c-4.7-4.7-4.7-12.3 0-17l65.6-65-65.6-65.1c-4.7-4.7-4.7-12.3 0-17l39.6-39.6c4.7-4.7 12.3-4.7 17 0l65 65.7 65.1-65.6c4.7-4.7 12.3-4.7 17 0l39.6 39.6c4.7 4.7 4.7 12.3 0 17L312 256l65.6 65.1z"/></svg> The community diversity estimate is limited

]

???

- **Easy Poll**

- Focalizziamo la nostra attenzione sul sistema coltura dipendente

- Questo approccio ha un grande punto di forza: Permette di isolare i microbi in coltura pura (ovviamente tutti quelli coltivabili in vitro - esclusi i simbioti obbligati)

- Il problema di questo approccio è sicuramente la sottostima della diversità all'interno del microbiota
]
---

]

.pull-right[
<img src="images/microbiotaCompositionTecniques.png" width="520px" style="display: block; margin: auto;" />
]

???

.h201[
- Il metodo indipendente dalla coltura, che approfondiremo meglio nelle prossime slides, ha 2 indiscutibili vantaggi: 
  - L'abilità non solo di identificare ma anche di quantificare l'abbondanza dei microorganismi presenti. Permettendo di identificare anche organismi estremamente poco abbondanti     
  - Utilizzando approcci bioinformatici è possibile ricavare importati informazioni sul funzionamento dell'intera comunità e non solo sui singoli individui
  
- Questo approccio, però, non permette di isolare i singoli microrganismi

- Isolare i singoli microorganismi può essere importante per la riscostruzione delle Comunità Sintetiche che permettono lo studio in laboratorio o l'applicazione in agricoltura per migliorare la resa delle colture vegetali
]

---

> **DNA metabarcoding** is a method of **DNA barcoding** that uses universal genetic markers to identify DNA of a mixture of organisms

???

- Vediamo ora come studiare il microbiota con **tecniche coltura indipendenti**!
- Per studiare il microbiota e quindi identificare gli organismi lo compongono si utilizza una tecnica chiamata **DNA METABARCODING**

- Questa tecnica utilizza il **DNA barcoding** per studiare la composizione del microbiota

- **CHE COS'E' il DNA BARCODING"**
]

<br>
.content-box-grey[What is it the <br> **DNA barcoding**?]

---

- **DNA barcoding** is a method of species identification using a short section of DNA from a specific gene

- An individual sequence can be used to uniquely identify an organism by comparison with a reference library of classified DNA sequences

???

.h201[
- Il DNA Barcoding è una tecnica che permette di identificare l'organismo studiato basandosi su una sua specifica sequenza nucleotidica

- L'idea è utilizzare una sequenza di DNA per identificare un organismo in maniera univoca.

- Dopo aver identificato la sequenza specifica, questa viene confrontata con un database di riferimento per essere classificata

- Un paragone potrebbe essere quello di utilizzare il vostro codica fiscale per identiifcarvi. La sequenza di lettere e numeri non mi da informazioni, ma cercandolo in un database potrei conosce il vostro nome e cognome  e identificarvi in modo univoco
]

---

- Ideally, one gene sequence would be used for developing universal PCR primers for all taxonomic group
  
- However, no such gene region has been found yet!

- Different gene regions are used to identify the different organismal groups using barcoding
]
]

???

.h201[
- Siccome i microrganismi non sono registrati all'anagrafe e non hanno un loro CF, cosa possiamo usare come sequenza univoca per identificarli?

- La selezione di queste sequenze marker (**chiamate barcode**) è cruciale per l'identificazione dei microorganismi

- In un modo perfetto esisterebbe un unico barcode, cioè un singolo marker che ci permeterebbe si identificare e distinguere tutti i gruppi tassonomici

- Ma ovviamente non viviamo in un mondo ideale! E questo marker non è ancora stato individuato e probabilmente non lo sarà mai

- Quindi, diversi gruppi tassonomici usano diversi tipi di barcodes

- Sulla destra della slide vedete un tipico cromatogramma di sequenziamento. Da quest'immagine è facile capire perchè i markers sono chiamati barcodes
]

---

**Sanger Sequencing**

.pull-left-narrow[
.h25[
<br><br>
Sanger sequencing, also known as the “chain termination method”, is a method for determining the nucleotide sequence of DNA. The method was developed by two time Nobel Laureate Frederick Sanger and his colleagues in 1977, hence the name the Sanger Sequence
]
]

.pull-right-wide[
<img src="images/sanger.png" width="820px" style="display: block; margin: auto;" />
]

---

**Marker selection**

| Organism | Region |
|----------|--------|
| animals  | cytochrome c oxidase I|
| fungi    |  internal transcribed spacer (ITS) rRNA |
| plants   | RuBisCO |
| prokaryotes | 16S rRNA |
| microbial eukaryotes | 18S rRNA |

.font80[When barcoding is used to identify organisms from a sample containing DNA from more than one organism, the term DNA metabarcoding is used]

???

- Questi geni sono stati scelti a causa della dell'evoluzione lenta

- COX1 enzima chiave del metabolismo aerobico

- Internal transcribed spacer (ITS): sono regioni non codificanti di DNA situate tra geni. ITS in partcilare tra le subunità dellRNA ribosomiale

- ribulosio-1,5-bisfosfato carbossilasi/ossigenasi: tramite la fissazione del carbonio, è il principale produttore di materia organica nell'ecosistema terrestre

- RNA ribosomiale 16 S (o rRNA 16 S) è una componente della subunità 30S di un ribosoma procariotico

- Il 18S è l'omologo eucariota del 16S dei procariotu
]

---

**Metabarcoding process**

???

---

.pull-left[
.m0p[
**Bioinformatic analysis**
]
.h201[
- After sequencing, raw data must be processed (**quality and cleaning step**)

- Samples are clustered in operational taxonomic unit based on identity threshold (**OTU formation**)

- Using "the Basic Local Alignment Search Tool (BLAST)" regions of similarity between sequences and reference databases are identified (**annotation step**)

- Microbiota composition definition (**richness and diversity step**)

]
]

.h20[
.m0p[
.right[.font50[(Jesse et al., 2018)]]     
]
<img src="images/OTU.png" width="220px" style="display: block; margin: auto;" />

]]

???

- An operational taxonomic unit (OTU) is an operational definition used to classify groups of closely related individuals
]

---
layout: true

# What can the microbiota potentially do?

---

----

???

.h201[
- Prendiamo ora in considerazione quella tecnica che oltre a permetterci di identificare i componenti del microbiota ci permette anche di capire cosa "potenzialmente" possono fare!
]
---

<img src="images/metagenomics1.png" width="720px" style="display: block; margin: auto;" />
.right[.font50[(Ye, Siddle, Park, and Sabeti, 2019)]]
.h25[
- **Metagenomics** is the study of genetic material recovered directly from environmental samples

- The collection of sequenced genes from the environment could be analyzed as a **single genome**
  
- Metagenomics **does not require the isolation** and lab cultivation of individual species

- Due to its random nature, metagenomics, allows that many **under-represented organisms will be detected**
]

???

- Questo campione può essere considerato come un singolo campione e quindi come tale analizzato

- La figura illustra la procedura di analisi di un campione di metagenomica. Come potete vedere 3 steps sono necessari:
  1. Estrazione
  1. Sequenziamento
  1. Classificazione
]

---

.center[Metagenomics vs Metabarcoding]
.pull-left[
.m0p[
<img src="images/compareBarcodingMetagenome.png" width="450px" style="display: block; margin: auto;" />
.right[.font50[(Gill, Baldock, Brown, Cresswell, Dicks, Fountain, Garratt, Gough, Heard, Holland, Ollerton, Stone, Tang, Vanbergen, Vogler, Woodward, Arce, Boatman, Brand-Hardy, and Potts, 2016)]]
]]

- Metagenomics does not need the marker selection step

- Metagenomics sequences all the genes present in the sample from each organism

- Metagenomics can help to reconstruct large fragments or even complete genomes from organisms in a community

- Metagenomics allows the characterization of a large number of coding and non-coding sequences that can be used to decipher the microbial diversity or to understand its metabolic potential
]
]

???

.h201[
- La differenza fondamentale tra Metagenomica e Metabarcoding è che per la Metagenomica non serve alcun barcode (marker)

- Questo perchè in metagenomica tutto il DNA presente nel campione viene sequenziato

- Il risultato del sequenziamento permette di ricostruire gran parte dei geni presenti nel microbiota, permettondo così di capire quali organismi sono presenti ma anche cosa potenzialmente questi geni possono esprimere
]

---

???

---

.center[
.middle[
<iframe width="840" height="473" src="https://www.youtube.com/embed/fCd6B5HRaZ8" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture" allowfullscreen></iframe>
]
https://bit.ly/2IGGiJE
]
---

???

.h201[
- I frammenti generati dal sequenziamento devono poi essere **assemblati** per ricostruire il/i genomi presenti nel campione

- 2 tipi assembli a seconda che sia presente un genoma di riferimento

- Mappaggio per dire chi è più o meno abbondante
]

---
.font120[**Functional Metagenomics Analysis**]

- Binning

- Metagenomic assembly

- Gene prediction

- Gene annotation

- Reconstruction of metabolic pathways (achieved from enzyme-coding genes)
]]

.pull-right[
<img src="images/metagenomicsPipeline.png" width="380px" style="display: block; margin: auto;" />
.right[.font50[(Roumpeka, Wallace, Escalettes, Fotheringham, and Watson, 2017)]]
]

???

- In questa slide vediamo come facciamo a definire quali sono i potenziali processi del microbiota analizzato

- In metagenomica, il binning è il processo di raggruppamento delle reads o contigs e di assegnarli a unità tassonomiche operative
]

---

- Binning

- Metagenomic assembly

- Gene prediction

- Gene annotation

- Reconstruction of metabolic pathways (achieved from enzyme-coding genes)
]]

.pull-right[
<img src="images/functionalMetagenomics.png" width="370px" style="display: block; margin: auto;" />
.right[.font50[(Castañeda and Barbosa, 2017)]]
]

---
layout: true

# What are they doing?

---

----

???

<img src="images/Meta1.png" width="620px" style="display: block; margin: auto;" />
.right[.font50[(Vanwonterghem, Jensen, Ho, Batstone, and Tyson, 2014)]]

???

- Abbiamo visto come il DNA Metabercoding risponda alla domanda "Chi è presente?"

- Il Metagenoma risponde sia alla domanda "Chi è presente?" sia alla domanda "Cosa potenzialmente puà fare il microbiota"

- Vediamo ora quali tecniche ci permettono di studiare le funzioni del microbiota:

1. Metatranscriptomics
1. Metaproteomics
1. Metabolomics
1. SIP: Stable isotop probing
]

---

Metatranscriptomics

<img src="images/Meta2.png" width="620px" style="display: block; margin: auto;" />
.right[.font50[(Vanwonterghem, Jensen, Ho, et al., 2014)]]

---

Metatranscriptomics
.pull-left[
.h25[
- Metatranscriptomics is the science that studies gene expression of microbes within natural environments

- It allows to obtain whole gene expression profiling of complex microbial communities

-  The overview of the gene expression in a given sample is obtained by capturing the total mRNA of the microbiome and by performing a whole metatranscriptomics shotgun sequencing.
]
]

.pull-right[
.m0p[
<img src="images/metatrranscriptomePL.png" width="380px" style="display: block; margin: auto;" />
.right[.font50[(Pérez-Pantoja and Tamames, 2015)]]
]
]

???

.h201[
- La metatranscrittomica studia l'espressione genica dei microorganismi nell'ambiente ovvero studia l'**mRNA**

- Essendo una tecnica basata sul sequenziamento permette di ottenere tutti gi mRNA messaggeri presenti nel campione e quindi di avere il profili di espressione dell'ologenoma

- Guardando la figura salla dx vediamo come si sviluppa schematicamente un'analisi di metatrascrittomica

- [...]

- Mi permetto di farvi notate la diversa dimensione dei 2 riquadri e dell'importanza che riveste oggi la parte di bioinformatica
]
---

Metaproteomics

<img src="images/Meta3.png" width="620px" style="display: block; margin: auto;" />
.right[.font50[(Vanwonterghem, Jensen, Ho, et al., 2014)]]

---

Metaproteomics

.pull-left[
.h25[
- Metaproteomics is the study of all protein samples recovered directly from environmental sources

- Metaproteomics have rapidly increased in recent years due to many technological advances in mass spectrometry (MS)

.pull.right[
<img src="images/metaproteomics.png" width="480px" style="display: block; margin: auto;" />
.right[.font30[https://www.ecu.edu.au/schools/science/research-activity/research-support/proteomics/what-is-lc-ms-proteomics]]
]

???

.h201[
- La Metaproteomica studia le proteine espresse dalla comunità di microorganismi presa in esame

- La proteomics lavora sulle proteine espresse, cioè i veri attori del metabolismo. A differenza di quanto fa la trascrittomica

- Come funziona la proteomica
]

---

Metabolomics

<img src="images/Meta4.png" width="600px" style="display: block; margin: auto;" />
.right[.font50[(Vanwonterghem, Jensen, Ho, et al., 2014)]]

---

Metabolomics

- Metabolomics is the study of the complete set of metabolites composition (the metabolome) of a cell type, tissue, or biological fluid

.pull-rigth[
<br><br>
<img src="images/metabolomics.png" width="550px" style="display: block; margin: auto;" />
]
.right[.font70[(Alonso, Marsal, and Julià, 2015)]]

???

- I metaboliti sono molecole di dimensione inferiore ai 1.5 KDa e rappresentano gli intermedi o i prodotti finali delle reazioni enzimatiche

- Ci informano per tanto sulle reali attività biochimiche in atto!!

- Esempi di metaboliti sono: carboidrati idrofilici, alcool volatili, chetoni, metaboliti secondari come antibiotici e pigmenti

- Descrizione figura

- Spettroscopia di risonanza magnetica nucleare
]

---

Stable/radioactive isotopes

<img src="images/Meta5.png" width="600px" style="display: block; margin: auto;" />
.right[.font50[(Vanwonterghem, Jensen, Ho, et al., 2014)]]

---

Stable/radioactive isotopes

- Stable-isotope probing (SIP) is a technique in microbial ecology for tracing fluxes of nutrients in biogeochemical cycling by microorganisms using heavy stable isotopes

.pull-rigth[
<img src="images/sip1.png" width="280px" style="display: block; margin: auto;" />
]
.right[.font50[(Uhlik, Leewis, Strejcek, Musilova, Mackova, Leigh, and Macek, 2013)]]

???

.h201[
- La Stable isotope probing è una tecnica che utilizzando isotopi radioattivi traccia il flusso dei nutrienti utilizzati dai microorganismi

- Tra i pro di questa tecnica c'è la possibilità di studiare in detttaglio specifiche funzioni della popolazione microbica

- Per contro questa tecnica è molto complessa a livello pratico e molto costaosa a livello economico
]

---

Stable/radioactive isotopes

.m0p[
<img src="images/sip2.png" width="570px" style="display: block; margin: auto;" />
.right[.font50[(el Zahar Haichar, Heulin, Guyonnet, et al., 2016)]]
]

---
layout: false
class: clear, middle, center

???
metafora della torta e dei suoi ingredienti

---
class: clear

.pull-left[
.m0p[
.h25u[
- .font80[Systems biology studies biological systems by systematically perturbing them]

- .font80[Monitoring the gene, protein, metabolite, and informational pathway responses]

- .font80[Integrating these data]

- .font80[Formulating mathematical models that describe the structure of the system]

- .font80[Predict its response to individual perturbations]
  ]]]

]
]

???

- La soluzione migliore è usarle tutte e combinarle insieme in quello che viene chiamato System biology

- Tutti questi dati integrati dovrebbero permetterci una migliore comprensione del sistema studiato, aiutarci a create dei modelli e grazie a questi modelli predirre le risposte del sistema
]

---

# Questions about the lesson

---

.m0tbp[
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]