Lecture 1

Log in using ssh

ssh user@workshop2021a.vhost37.genap.ca

Create a directory with mkdir

mkdir -p ~/projects/lec1

Go to the created directory with cd

cd ~/projects/lec1

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Copy over files with cp

cp -R /home/hgen_share/lec1/* ./

Show the list of files with ls

ls -lh

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Inspect the first few lines of ex* files with head

head ex1.fa
head ex2.fq

Inspect the seqtk module with module spider

module spider seqtk/1.3

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Load the seqtk module with module load

module load StdEnv/2020 seqtk/1.3

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Inspect loaded modules with module list

module list

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Convert FASTQ to FASTA with seqtk seq

seqtk seq -a ex2.fq > ex2.fa

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Inspect the first few lines of ex2.fa with head

head ex2.fa

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Export conda environment

export PATH="/home/hgen_share/Anaconda/bin:$PATH"
 
export PATH="/home/hgen_share/Anaconda/envs/lec1/bin:$PATH"

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Check environment is in your path

echo $PATH

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More information on how to set up Anaconda environment here

Perform quality control analysis on ex2.fq with fastqc

fastqc ex2.fq

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Download results to your local computer using scp

scp user@workshop2021a.vhost37.genap.ca:/directory/fastqc_results.html local_directory 

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Inspect the reference assembly

head /home/hgen_share/bwa/hg19.fa

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Assign the reference file to a variable for simplicity

ref=/home/hgen_share/bwa

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Subset the first 10 and then 1000 sequences of ex2.fa using seqkit

seqkit head -n 10 ex2.fa > sub_ex2.fa

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Time the alignment against hg19 with blat using a command of the form

module load blat/3.5
time blat $ref/hg19.fa sub_ex2.fa OUTPUT.psl

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More information on the .psl format found here

Align against the same reference with bwa mem

module load bwa/0.7.17
bwa mem $ref/hg19.fa ex2.fq > ex2.sam

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How many reads are there in ex2.fq? How much faster is BWA MEM compared to BLAT?

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Take a look at the *.sam file with less

less ex2.sam

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Calculate summary statistics with samtools flagstat

module load samtools/1.16.1
samtools flagstat ex2.sam

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More information on interpreting flagstat statistics found here

Sort and compress in one go with samtools sort

samtools sort ex2.sam > ex2.sorted.bam 

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Take a look at the *.bam file with head

head ex2.sorted.bam

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Compare the size difference after compression with ls

ls -lh ex2.*am

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Generate an index file with samtools index

samtools index ex2.sorted.bam