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# Starting a Metagenomics Project

## Overview

Teaching: 15 min
Exercises: 15 min
Questions
• How do you plan a metagenomics experiment?

• How does a metagenomics project look like?

Objectives
• Learn the differences between shotgun and metabarcoding (amplicon metagenomics) techniques.

• Understand the importance of metadata.

• Familiarize yourself with the Cuatro Ciénegas experiment.

## Metagenomics

Metagenomes are collections of genomic sequences from various (micro)organisms that coexist in any given space. They are like snapshots that can give us information about the taxonomic and even metabolic or functional composition of the communities that we decide to study. Thus, metagenomes are usually employed to investigate the ecology of defining characteristics of niches (e.g., the human gut, or the ocean floor).

Since metagenomes are mixtures of sequences that belong to different species, a metagenomic workflow is designed to answer two questions:

1. What species are represented in the sample?
2. What are they capable of doing?

To find which species are present in a niche, we have to do a taxonomic assignation of the obtained sequences. To find out their capabilities, we can look at the genes directly encoded in the metagenome or the genes associated with the species that we found. In order to know which methodology we should use, it is important to know what questions do we want to answer.

## Shotgun and amplicon

There are two paths to obtain information from a complex sample:

1. Shotgun Metagenomics
2. Metabarcoding.

Each is named after the sequencing methodology employed and have particular use cases, with inherent advantages and disadvantages.

With Shotgun Metagenomics, we sequence random parts (ideally all of them) of the genomes present in a sample. We can search the origin of these pieces (i.e., their taxonomy) and also try to find to what part of the genome they correspond. Given enough pieces, it is even possible to obtain full individual genomes from a shotgun metagenome, which could give us a bunch of information about the species in our study. This, however, requires that we have a lot of genomic sequences from one organism, and since the sequencing is done at random, we usually have to sequence our community a lot (have a high sequencing depth) to make sure that we obtain enough pieces of a given genome. This gets exponentially harder when our species of interest is not very abundant. It also requires that we have enough DNA to work with, which can be difficult to obtain in certain cases. Finally, a lot of sequencing means a lot of expenses, and because of this, making technical and biological replicates can be prohibitively costly.

On the contrary, Metabarcoding tends to be cheaper, which makes it easier to duplicate and even triplicate them without taking a big financial hit. This is because Metabarcoding is the collection of small genomic fragments present in the community and amplified through PCR. If the amplified region is present only once in every genome, ideally, we wouldn’t need to sequence the amplicon metagenome so thoroughly because one sequence is all we need to get the information about that genome, and by extension, about that species. On the other hand, if a genome in the community lacks the region targeted by the PCR primers, then no amount of sequencing can give us information about that genome. This is why the most popular amplicon used for this methodology are 16S amplicons for Bacteria since every known bacterium has this particular region. Other regions can be chosen, but they are used for very specific cases. However, even 16S amplicons are limited to, well, the 16S region, so amplicon metagenomes cannot directly tell us a lot about the metabolic functions found in each genome, although educated guesses can be made by knowing which genes are commonly found in every identified species.

Once we have chosen an adequate type of methodology for our study, it is important to take extensive notes on the origin of our samples and how we treated them. These notes constitute the metadata, or data about our data, and it is crucial to understand and interpret the results that we are going to obtain later on in our metagenomic analysis. Most of the time, the differences that we observe when comparing metagenomes can be correlated to the metadata, which is why we must include a whole section of our experimental design to the metadata that we expect to collect and record carefully.

## Amplicon or Shotgun?

Suppose you would like to compare the gut microbiome of people affected by a rather nasty bacterial disease against the gut microbiome of healthy people.
Which type of metagenomics would you choose?
Which type of metadata would be useful to record?

Before we continue we want to introduce you Chepiche; they are going to be with us during this lesson because they are also interested in learning about metagenomics, in fact they already have Cuatro Ciénegas data to work on! Let’s see!

## Cuatro Ciénegas

During this lesson, we will work with actual metagenomic information, so we should be familiarized with it. The metagenomes that we will use were collected in Cuatro Ciénegas, a region that has been extensively studied by Valeria Souza. Cuatro Ciénegas is an oasis in the Mexican desert whose environmental conditions are often linked to the ones present in ancient seas, due to a higher than average content of sulfur and magnesium but lower concentrations of phosphorus and other nutrients. Because of these particular conditions, the Cuatro Ciénegas basin is a very interesting place to conduct a metagenomic study, to learn more about the bacterial diversity that is capable to survive and thrive in that environment.

The particular metagenomic study that we are going to work with was collected in a study about the response of the Cuatro Cienegas’ bacterial community to nutrient enrichment. In this study, authors compared the differences between the microbial community in its natural, oligotrophic, phosphorus-deficient environment, a pond from the Cuatro Ciénegas Basin (CCB), and the same microbial community under a fertilization treatment. The comparison between bacterial communities showed that many genomic traits, such as mean bacterial genome size, GC content, total number of tRNA genes, total number of rRNA genes, and codon usage bias were significantly changed when the bacterial community underwent the treatment.

According to the results described for this CCB study.

1. What kind of sequencing method do you think they used, and why do you think so?
A) Metabarcoding B) Shotgun metagenomics
C) Genomics of axenic cultures

2. In the table samples treatment information, what was the most important piece of metadata that the authors took?

## Solution

A) Metabarcoding. False. With this technique, usually only one region of the genome is amplified.
B) Shotgun Metagenomics. True. Only shotgun metagenomics could have been used to investigate the total number of tRNA genes.
C) Genomics of axenic cultures. False. Information on the microbial community cannot be fully obtained with axenic cultures.

The most important thing to know about our data is which community was and was not supplemented with fertilizers.
However, any differences in the technical parts of the study, such as the DNA extraction protocol, could have affected the results, so tracking those is also important.

## Exercise 2: Differentiate between IDs and sample names

Depending on the database, several IDs can be used for the same sample. Please, open Chepiche’s document where the metadata information is stored. Here inspect the IDs and find out which of them correspond to sample JP4110514WATERRESIZE

## Solution

ERS1949771 is the SRA ID corresponding to JP4110514WATERRESIZE

## Exercise 3: Discuss the importance of Metadata

Which other data could you recommend Chepiche to add in their data, and what did you think is the relevance of this information?

## Solution

Metadata will depend on the type of the experiment, but some examples are temperature, sampling methodology, date, place (country, state, region, city, etc.).

Note that throughout the lesson, we will use the first four characters of the file names (alias) to identify the data files corresponding to a sample.

The results of this study, raw sequences, and metadata have been submitted to the NCBI Sequence Read Archive (SRA), and are stored in the BioProject PRJEB22811. There are other metagenomic databases where we can find metagenomics data.

## Other metagenomic databases

The NCBI SRA is not the only repository for metagenomic information. There are other public metagenomic databases such as MG-RAST, MGnify, Marine Metagenomics Portal, Terrestrial Metagenome DB and the GM Repo.

Each database requires certain Metadata linked with the data. As an example, when JP4D.fasta is uploaded to mg-RAST the associated Metadata looks like:

Column Description
file_name JP4D.fasta
investigation_type metagenome
seq_meth illumina
project_description This project is a teaching project and uses data from Okie et al Elife 2020
collection_date 2012-06-27
country Mexico
feature pond water
latitude 26.8717055555556
longitude -102.14
env_package water
depth 0.165

## Key Points

• Shotgun metagenomics can be used for taxonomic and functional studies.

• Metabarcoding can be used for taxonomic studies.

• Collecting metadata beforehand is fundamental for downstream analysis.

• We will use data from a Cuatro Ciénegas project to learn about shotgun metagenomics.