Getting Error In Bowtie2

Question

[deepthithomaskannan]

Hi all,

I tried the below bowtie2 command: bowtie2 -q --phred33 -x targreads -U <input_fastq_file> -S <output_sam> --un <output_unaligned_fastq> --no-unal --rg-id <rgid> --rg <sm:sample_name>

I am getting the following error: Use of uninitialized value in scalar chomp at /export/data/linux/bowtie2/bowtie2-2.0.4/bowtie2 line 408, <bt> line 4387707. Use of uninitialized value in substitution (s///) at /export/data/linux/bowtie2/bowtie2-2.0.4/bowtie2 line 409, <bt> line 4387707.

Anybody know how to resolve this? Is there any problem with my parameters?

Thanks, Deepthi

Answer 1

[matted]

Yes, there are problems. You are missing filenames or strings as arguments for --un, -S, -U, --rg-id, and --rg. Read the documentation, and in particular the getting started section, and try again.

[deepthithomaskannan]

Actually I added file names. Editor deleted it. I am re entering the command. bowtie2 -q --phred33 -x targreads -U In_file.fastq -S out_file.sam --un out_file_unaligned.fastq --no-unal --rg-id RGID --rg SM:sample_name

Answer 2

[skymningen]

I think there are several problems. First, like matted said, you miss some parameter values. Second, I am not sure you got the right input for -x as well.

So there's what you did:

bowtie2 -q --phred33 -x targreads -U -S --un --no-unal --rg-id --rg

I'll be speculating on some things you might have/want now. I guess you have some input fastq of (unpaired) reads you want to align (in.fq) . And I hope you also have a reference in fast format that you want them to be aligned to (ref.fasta). Then the first thing you are supposed to do is build an index from the reference. Not from the reads, which it looks like you have done because you used "targreads" for -x.

bowtie2-build -f ref.fasta reference

This will give you an index of the reference called "reference", which is what you need for -x

Now a basic bowtie2 command would look like this:

bowtie2 -x reference -U in.fq -S output.sam

You don't need to put in the -q, it's default. If you input reads are fasta, you need to give it -f.

bowtie2 -f -x reference -U in.fasta -S output.sam

If they are paired fastq, you need to give both files.

bowtie2 -x reference -1 in1.fq -2 in2.fq -S output.sam

Those are the basics. All the other commands depend on what you have and what you want with it, you should reference to the manual for those.

[deepthithomaskannan]

I already built index for reference reads. Actually I have done all the steps you described. Actually I added file names in my command. Editor deleted it. I did not notice. sorry about that.

The command I used is bowtie2 -q --phred33 -x targreads -U In_file.fastq -S out_file.sam --un out_file_unaligned.fastq --no-unal --rg-id RGID --rg SM:sample_name

[skymningen]

Did you check if it runs with just bowtie2 -q --phred33 -x targreads -U In_file.fastq -S out_file.sam ? Then all you need is adding the other parameters one by one and check from what parameter your error comes from.