to run bowtie on multiple fastq file

Question

[Kritika]

Hello all

Currently I am running bowtie to align my 4 pairend reads to reference genome . I am running shell script but it's showing error

for I in $(path_to_my_fastqfiles/*.fastq)
do
    /opt/bowtie2-2.2.6/bowtie2 -p4 -x path_to_my_reference genome/ref/  path_to_my_fastqfile -1 ${i}.fastq -2 ${i}.fastq -S $i.sam
done

I am getting error permission denied. Even if I do with sudo still it's showing same error.

also if I change the first line for I in $(ls *.fastq), I get this error

Warning: Same mate file "ls" appears as argument to both -1 and -2
Extra parameter(s) specified: "path_to_my_fastqfiles", "*.fastq", "*.fastq", "*.fastq.sam"
Note that if <mates> files are specified using -1/-2, a <singles> file cannot
also be specified.  Please run bowtie separately for mates and singles.
Error: Encountered internal Bowtie 2 exception (#1)
Command: /opt/bowtie2-2.2.6/bowtie2-align-s --wrapper basic-0 -p4 -x /path_to_my_ref/ -S ls -1 ls -2 ls path_to_my_fastqfile  *.fastq *.fastq *.fastq.sam 
(ERR): bowtie2-align exited with value 1

[GouthamAtla]

Can you post exact error ? Permission denied to read the files or execute the command or create sam file ?

Irrespective of permissions error, I do not know why it should work if you use -1 ${i}.fastq -2 ${i}.fastq essentially both R1 and R2 are same files ?

check bash loop for alignment RNA-seq data

[Kritika]

I changed my script : -1 ${i}A_R1.fasta -2 ${i}A_R2.fasta

My files are 1A_R1.fastq 1A_R2.fastq 2A_R1.fastq 2A_R2.fastq ....

/media/74BE94C7BE948372/sample/to_run_all_file.sh: 1: /media/74BE94C7BE948372/sample/to_run_all_file.sh: /media/74BE94C7BE948372/sample/fastq_file/1A_R1.fastq: Permission denied

[GouthamAtla]

It should be pretty simple if you exactly show us how your files are named. can you show us the output of the command ls path_to_my_fastqfiles/*.fastq

[Kritika]

1A_R1.fastq 2A_R1.fastq 3A_R1.fastq 4A_R1.fastq
1A_R2.fastq 2A_R2.fastq 3A_R2.fastq 4A_R2.fastq

this are my file names this are stored in media/sample/fastqfiles folder

[John]

for i in $(path_to_my_fastqfiles/*.fastq) is going to return the full path of your fastqs, so $iA_R2.fasta will become /path/to/1A_R1.fastqA_R1.fasta

This is not what you wanted I guess?

First off - are you using fasta or fastq because you change between the two...? Second off (is that a thing?) - you want to target ONLY THE R1s when assigning $i, and then cut off the last 8 characters. Do that with:

for i in $(path_to_my_fastqfiles/*R1.fastq)
do
    yolo=$(echo "$i" | rev | cut -c 9- | rev) # dumb way of cutting off the last 8 chars
    /opt/bowtie2-2.2.6/bowtie2 -p4 -x path_to_my_reference genome/ref/ -1 {$yolo}R1.fastq -2 {$yolo}R2.fastq -S {$yolo}.sam
done

Also I suck at bash so theres a good chance you need to quote things or remove those {brackets} or something, but try it and see :)

Also also, this is why i hate defining what to run and running it at the same time. Although others may disagree, you should write a script that generates a list of commands to execute, so 1) you have it on record, 2) you can see what your doing as you iteratively create the list of commands to run.

[Kritika]

sorry its fastq file only showing same error

[John]

OK. Did you try the script above?

[John]

If you paste the full filepath of both your -1 and -2, we can help you write a bash loop that works :)

[Joe]

Dont surround the path with $(). That is a process substitution and the shell will try and execute the file path as a command.

If you need a list of files try:

for i in path/to/file/*.fastq ;

or

for i in $(ls /path/to/files) ;

Answer 1

[GouthamAtla]

There are multiple ways but this should also work. You can specify a output directory where you want to save sam files.

for sample in `ls /media/sample/fastqfiles/*R1.fastq`
do
dir="/media/sample/fastqfiles"
base=$(basename $sample "_R1.fastq")
bowtie2 -x path_to_my_index -1 ${dir}/${base}_R1.fastq -2 ${dir}/${base}_R2.fastq -S ${dir}/${base}.sam
done

Always crosscheck what is doing with echo statement.

echo "bowtie2 -x path_to_my_index -1 ${dir}/${base}_R1.fastq -2 ${dir}/${base}_R2.fastq -S ${dir}/${base}.sam"

[John]

Oooh, basename takes a second parameter? Awesome :D Thanks Goutham :)

Answer 2

[surendra]

Hi

You can run the bowtie2 with the following command

bowtie2 -x refernce_file -1 Forward_read_file_A_1, Forward_read_file_B_1 -2 Reverse_read_file_A_2, Reverse_read_file_B_2

I think it will solve your problem.