Lab07

Now activate the environment you created last week.

conda activate bfblab

Install the tools that we will use

#Install FASTQC for sequence quality check
conda install -c bioconda fastqc

#Install fastp for FASTQ trimming
conda install -c bioconda fastp

#Install Megahit for de novo assembly
conda install -c bioconda megahit
#Then, this will show the user manual:
megahit -h

#Install QUAST for fasta quality check
conda install -c bioconda quast

#Install gdown for file downloading
conda install -c conda-forge gdown

#or you can install all of them at once
conda install -c bioconda -c conda-forge fastqc fastp megahit quast gdown

conda create --name newenv
conda activate newenv

conda install -c bioconda -c conda-forge quast gdown

Obtain the sequencing reads and check their quality

First, create a separate folder for the lab exercises:

cd ~

mkdir Lab07

cd Lab07

Download the data using this link using wget:

#Download the first read:
gdown https://drive.google.com/uc?id=1CiRkrUcP3S_oNiluGQ1Mh1qSU-3FpKdF

#Download the second read:
gdown https://drive.google.com/uc?id=14di4CJ_J8TrRwISRQm0Nbt9_tILcYF9N

Open and visualize the sequencing reads in one of the fastq files. Pay attention to using ‘|’ (pipe)!

gunzip -c reads_1.fq.gz | less

Check the quality of sequences: Run the FASTQC software on both read_1 and read_2 files as follows:

conda activate bfblab

# *.gz means that select any file which ends with ".gz" ( these characters are called wildcards )
fastqc *.gz

Open the html outputs generated using a web browser. Investigate the results.

Trimming FASTQ

Trimming/cleaning the data may be required after QC analysis

#Trimming using fastp (paired-end) (accepts both .fq and fq.gz)
fastp -i reads_1.fq.gz -I reads_2.fq.gz -o out.reads_1.fq.gz -O out.reads_2.fq.gz
#for single read
fastp -i read.fq -o out.read.fq

De Novo Genome assembly using MegaHit

After quality check and trimming(if required) we may now assemble the reads into contigs

megahit -1 out.reads_1.fq.gz -2 out.reads_2.fq.gz -o Unknown_genome_megahit

This command will create a folder called ‘Unknown_genome_megahit’. Please, navigate into that folder.

cd Unknown_genome_megahit/

ls -al

#And open the final contigs fasta file
less final.contigs.fa

Questions to answer:

1. How many contigs did the assembly produce?

2. What is the length of the total genome size?

3. What is the coverage (C) of this genome?

4. Which organism does this genome belong to? How can you find out?

To answer this questions we can use QUAST to check the quality of the assembled genome (FASTA files)

#QUAST reads the fasta file and creates several statistical reports regarding that file
quast final.contigs.fa
#after opening the reports folder
less report.txt
#you can see and analyse the end report of QUAST and answer the previous questions

Example bash script to wrap up all the code above:

write these in a file using a text editor (gedit)

#!/usr/bin/bash

#set the arguments to variables
FASTQ_1=$1
FASTQ_2=$2

#Check the quality of input reads
fastqc $FASTQ_1

fastqc $FASTQ_2

#Trimming using fastp (paired-end) (accepts both .fq and fq.gz)
fastp -i $FASTQ_1 -I $FASTQ_2 -o trimmed."${FASTQ_1}" -O trimmed."${FASTQ_2}"

#Run de novo assembly using MegaHit
megahit -1 trimmed."${FASTQ_1}" -2 trimmed."${FASTQ_2}" -o Unknown_genome_megahit