QC/QA Hands on tutorial

Link to presentation

Data

Login

• Using putty/mobaxterm

• Command line using

  ssh username@115.146.84.253

List all the files, using command

ls

There should hopefully be a folder labelled “subsample”

If you have only “subsample.tar” folder, this means you have to decompress this file. To do this run the command

tar -xvf subsample.tar

If you need to download the files, follow the instructions here

List out the files in the subsample folder,

ls subsample

This should list the 8 metagenomes in the folder

SRR1237780_1.fastq.gz  SRR1237781_1.fastq.gz  SRR1237782_1.fastq.gz  SRR1237783_1.fastq.gz
SRR1237780_2.fastq.gz  SRR1237781_2.fastq.gz  SRR1237782_2.fastq.gz  SRR1237783_2.fastq.gz

Decompress the files by running the command

gzip -d SRR1237780_1.fastq.gz
gzip -d SRR1237780_2.fastq.gz
gzip -d SRR1237781_1.fastq.gz
gzip -d SRR1237781_2.fastq.gz
gzip -d SRR1237782_1.fastq.gz
gzip -d SRR1237782_2.fastq.gz
gzip -d SRR1237783_1.fastq.gz
gzip -d SRR1237783_2.fastq.gz

If the files are decompressed they should no longer have the .gz extension

FastQC

A quality control tool for high throughput sequence data - https://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Instead of downloading the application locally, we can run the application as commands. Run the below commands,

cd
mkdir fastqc
fastqc subsample/SRR1237780_1.fastq -o fastqc/

The FastQC report for this file is saved to the directory fastqc. Check this diretcory after the command is run

cd 
cd fastqc/
ls

This should list the below files

(base) nala0006@workshoppy:~/fastqc$ ls
SRR1237780_1_fastqc.html  SRR1237780_1_fastqc.zip

To visualize the output, copy the file to your laptop.

• This can be done using WinsSCP (https://winscp.net/eng/index.php)

• Command line

  scp -r nala0006@115.146.84.253:/home/nala0006/fastqc/SRR1237780_1_fastqc.html .
  

Once copied to laptop, click on the file and it should open in a browser

Repeat the same steps for reverse read, SRR1237780_2 Commands to run

fastqc subsample/SRR1237780_2.fastq -o fastqc/

Now there should be 4 files in fastqc

(base) nala0006@workshoppy:~$ ls fastqc/
SRR1237780_1_fastqc.html  SRR1237780_1_fastqc.zip  SRR1237780_2_fastqc.html  SRR1237780_2_fastqc.zip

Download SRR1237780_2_fastqc.html, and open in browser

Prinseq

Prinseq++ can be used to trim, filter and reformat sequences.

cd 
mkdir prinseq 

prinseq++ -min_len 60 -min_qual_mean 25 -ns_max_n 0 -derep 1  -out_format 0 -trim_tail_left 5 -trim_tail_right 5  -trim_qual_type min -trim_qual_left 30 -trim_qual_right 30 -trim_qual_window 10-threads 2 -out_name prinseq/SRR1237780 -fastq subsample/SRR1237780_1.fastq -fastq2 subsample/SRR1237780_2.fastq 

prinseq++ -min_len 60 -min_qual_mean 25 -ns_max_n 0 -derep 1  -out_format 0 -trim_tail_left 5 -trim_tail_right 5  -trim_qual_type min -trim_qual_left 30 -trim_qual_right 30 -trim_qual_window 10-threads 2 -out_name prinseq/SRR1237781 -fastq subsample/SRR1237781_1.fastq -fastq2 subsample/SRR1237781_2.fastq 

prinseq++ -min_len 60 -min_qual_mean 25 -ns_max_n 0 -derep 1  -out_format 0 -trim_tail_left 5 -trim_tail_right 5  -trim_qual_type min -trim_qual_left 30 -trim_qual_right 30 -trim_qual_window 10-threads 2 -out_name prinseq/SRR1237782 -fastq subsample/SRR1237782_1.fastq -fastq2 subsample/SRR1237782_2.fastq 

prinseq++ -min_len 60 -min_qual_mean 25 -ns_max_n 0 -derep 1  -out_format 0 -trim_tail_left 5 -trim_tail_right 5  -trim_qual_type min -trim_qual_left 30 -trim_qual_right 30 -trim_qual_window 10-threads 2 -out_name prinseq/SRR1237783 -fastq subsample/SRR1237783_1.fastq -fastq2 subsample/SRR1237783_2.fastq 

Output from prinseq would contain,

• good_out: filtered sequences for forward and reverse reads
• bad_out: removed sequences for forward and reverse reads

• single_out: singletons for forward and reverse reads

  (base) nala0006@workshoppy:~$ ls prinseq/
  SRR1237780_bad_out_R1.fastq     SRR1237781_good_out_R1.fastq    SRR1237782_single_out_R1.fastq
  SRR1237780_bad_out_R2.fastq     SRR1237781_good_out_R2.fastq    SRR1237782_single_out_R2.fastq
  SRR1237780_good_out_R1.fastq    SRR1237781_single_out_R1.fastq  SRR1237783_bad_out_R1.fastq
  SRR1237780_good_out_R2.fastq    SRR1237781_single_out_R2.fastq  SRR1237783_bad_out_R2.fastq
  SRR1237780_single_out_R1.fastq  SRR1237782_bad_out_R1.fastq     SRR1237783_good_out_R1.fastq
  SRR1237780_single_out_R2.fastq  SRR1237782_bad_out_R2.fastq     SRR1237783_good_out_R2.fastq
  SRR1237781_bad_out_R1.fastq     SRR1237782_good_out_R1.fastq    SRR1237783_single_out_R1.fastq
  SRR1237781_bad_out_R2.fastq     SRR1237782_good_out_R2.fastq    SRR1237783_single_out_R2.fastq
  

Yay!! All done with this tuorial too