Encountered internal Bowtie 2 exception (#1) bowtie2-align exited with value 1

Question

[luzglongoria]

Hi!

I am trying to assembly with bowtie2 and I got this errors. Here is my script:

bowtie2 --threads 4 --local --no-unal -x /home/luz_garcia_longoria/workspace/referencegenome/sturnus_relictumgenome.fasta -q -k 1 --al aligned_reads.fastq -U /home/luz_garcia_longoria/workspace/*.fasta > aligned_host_parasite.sam

stat: No such file or directory Warning: Could not open read file "/home/luz_garcia_longoria/workspace/*.fasta" for reading; skipping... Error: No input read files were valid (ERR): bowtie2-align exited with value 1 luz_garcia_longoria ~$ bowtie2 --threads 4 --local --no-unal
-x /home/luz_garcia_longoria/workspace/referencegenome/sturnus_relictumgenome.fasta
-q -k 1 --al aligned_reads.fq -U /home/luz_garcia_longoria/workspace/*.fq > aligned_host_parasite.sam Warning: Output file '/home/luz_garcia_longoria/workspace/s22_2.fq' was specified without -S.  This will not work in future Bowtie 2 versions.  Please use -S instead. Extra parameter(s) specified: "/home/luz_garcia_longoria/workspace/s23_1.fq", "/home/luz_garcia_longoria/workspace/s23_2.fq", "/home/luz_garcia_longoria/workspace/s24_2.fq", "/home/luz_garcia_longoria/workspace/s25_1.fq", "/home/luz_garcia_longoria/workspace/s25_2.fq", "/home/luz_garcia_longoria/workspace/s31_1.fq", "/home/luz_garcia_longoria/workspace/s31_2.fq", "/home/luz_garcia_longoria/workspace/s32_1.fq", "/home/luz_garcia_longoria/workspace/s32_2.fq", "/home/luz_garcia_longoria/workspace/s33_1.fq", "/home/luz_garcia_longoria/workspace/s34_1.fq", "/home/luz_garcia_longoria/workspace/s34_2.fq", "/home/luz_garcia_longoria/workspace/s35_1.fq", "/home/luz_garcia_longoria/workspace/s35_2.fq" Error: Encountered internal Bowtie 2 exception (#1) Command: /var/bin/bowtie2-2.3.1/bowtie2-align-s --wrapper basic-0 --threads 4
--local -x /home/luz_garcia_longoria/workspace/referencegenome/sturnus_relictumgenome.fasta
-q -k 1 --passthrough -U /home/luz_garcia_longoria/workspace/s22_1.fq /home/luz_garcia_longoria/workspace/s22_2.fq /home/luz_garcia_longoria/workspace/s23_1.fq /home/luz_garcia_longoria/workspace/s23_2.fq /home/luz_garcia_longoria/workspace/s24_2.fq /home/luz_garcia_longoria/workspace/s25_1.fq /home/luz_garcia_longoria/workspace/s25_2.fq /home/luz_garcia_longoria/workspace/s31_1.fq /home/luz_garcia_longoria/workspace/s31_2.fq /home/luz_garcia_longoria/workspace/s32_1.fq /home/luz_garcia_longoria/workspace/s32_2.fq /home/luz_garcia_longoria/workspace/s33_1.fq /home/luz_garcia_longoria/workspace/s34_1.fq /home/luz_garcia_longoria/workspace/s34_2.fq /home/luz_garcia_longoria/workspace/s35_1.fq /home/luz_garcia_longoria/workspace/s35_2.fq  (ERR): bowtie2-align exited with value 1

I have been reading and maybe the problem is with the -U option. Should I change it by -1 and -2? the thing is I'm quite new in bioinformatics and I am not sure how to specify which read is which with this command.

Thank you in advance

[Sej Modha]

Hi there,

Could you post your script here? It looks like some parameter that you may have used to pass file name is empty. In bowtie2 commands filenames are .fasta and .fq which should befilename.fasta or filename.fq

bowtie2 --threads 4 --local --no-unal -x /home/luz_garcia_longoria/workspace/referencegenome/sturnus_relictumgenome.fasta -q -k 1 --al aligned_reads.fastq -U /home/luz_garcia_longoria/workspace/.fasta bowtie2 --threads 4 --local --no-unal -x /home/luz_garcia_longoria/workspace/referencegenome/sturnus_relictumgenome.fasta -q -k 1 --al aligned_reads.fq -U /home/luz_garcia_longoria/workspace/*.fq

Also, refer to Bowtie2 manual to find out whether you should use -U (unpaired) or -1 and -2 (paired) parameters.

[luzglongoria]

Sorry, the code is this one. I wrote .fq not .fasta.

 bowtie2 --threads 4 --local --no-unal -x /home/luz_garcia_longoria/workspace/referencegenome/sturnus_relictumgenome.fasta -q -k 1 --al aligned_reads.fq -U /home/luz_garcia_longoria/workspace/*.fq > aligned_host_parasite.sam
Warning: Output file '/home/luz_garcia_longoria/workspace/s22_2.fq' was specified without -S.  This will not work in future Bowtie 2 versions.  Please use -S instead.
Extra parameter(s) specified: "/home/luz_garcia_longoria/workspace/s23_1.fq", "/home/luz_garcia_longoria/workspace/s23_2.fq", "/home/luz_garcia_longoria/workspace/s24_2.fq", "/home/luz_garcia_longoria/workspace/s25_1.fq", "/home/luz_garcia_longoria/workspace/s25_2.fq", "/home/luz_garcia_longoria/workspace/s31_1.fq", "/home/luz_garcia_longoria/workspace/s31_2.fq", "/home/luz_garcia_longoria/workspace/s32_1.fq", "/home/luz_garcia_longoria/workspace/s32_2.fq", "/home/luz_garcia_longoria/workspace/s33_1.fq", "/home/luz_garcia_longoria/workspace/s34_1.fq", "/home/luz_garcia_longoria/workspace/s34_2.fq", "/home/luz_garcia_longoria/workspace/s35_1.fq", "/home/luz_garcia_longoria/workspace/s35_2.fq"
Error: Encountered internal Bowtie 2 exception (#1)
Command: /var/bin/bowtie2-2.3.1/bowtie2-align-s --wrapper basic-0 --threads 4 --local -x /home/luz_garcia_longoria/workspace/referencegenome/sturnus_relictumgenome.fasta -q -k 1 --passthrough -U /home/luz_garcia_longoria/workspace/s22_1.fq /home/luz_garcia_longoria/workspace/s22_2.fq /home/luz_garcia_longoria/workspace/s23_1.fq /home/luz_garcia_longoria/workspace/s23_2.fq /home/luz_garcia_longoria/workspace/s24_2.fq /home/luz_garcia_longoria/workspace/s25_1.fq /home/luz_garcia_longoria/workspace/s25_2.fq /home/luz_garcia_longoria/workspace/s31_1.fq /home/luz_garcia_longoria/workspace/s31_2.fq /home/luz_garcia_longoria/workspace/s32_1.fq /home/luz_garcia_longoria/workspace/s32_2.fq /home/luz_garcia_longoria/workspace/s33_1.fq /home/luz_garcia_longoria/workspace/s34_1.fq /home/luz_garcia_longoria/workspace/s34_2.fq /home/luz_garcia_longoria/workspace/s35_1.fq /home/luz_garcia_longoria/workspace/s35_2.fq 
(ERR): bowtie2-align exited with value 1

[Sej Modha]

Are you trying to align all fastq files using this one command? It looks like you have paired-end data, are these meant to be paired-end files?

"/home/luz_garcia_longoria/workspace/s23_1.fq", "/home/luz_garcia_longoria/workspace/s23_2.fq"

The error that you are getting here is specifically related to missing -S parameter as you have not specified an output sam file.

I suggest that you should process each paired-end sample separately unless there is a specific reason why you'd want to combine all samples as unpaired data and align them to the reference genome.

[luzglongoria]

Thank you.

Actually there is no reason to do it like this. I thought I will save time :P

My data is paired-end data yes. I don't understand what do you mean with process each sample separately. You mean to run the same command with different data every time? Maybe I misunderstand something, sorry.

[Sej Modha]

I meant that individual commands should be run to process each sample separately. Please see http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#paired-inputs for more info. e.g. bowtie2 -x ref_index -1 file_1.fq -2 file_2.fq -S output.sam

On another note, why did you use rna-seq tag? Is this RNASeq data, if yes, then please use either HISAT2 or STAR instead of Bowtie2.

[luzglongoria]

It's RNA seq yes. What's the problem with bowtie2 (just curiosity)?

If I run the command you said and always enter the same output file, wouldn't delete what is inside and write the new results?

[GenoMax]

@Sej posted an example. You would not actually use the name output.sam for every pair of fastq files you have. You replace the relevant bits with your real file names for each set of PE reads.

Edit: It would be ok to use bowtie2 if this is bacterial data and you don't expect splicing to be a consideration. Otherwise use a splice-aware aligner as others have already noted.

[h.mon]

Bowtie2 is not designed to make spliced alignments, so it is not appropriate to map RNAseq to a genome reference - you should use STAR or HISAT2 instead, which are mappers that take into account spliced mappings.

If you are mapping to a reference transcriptome, then you can use Bowtie2.

[luzglongoria]

I am mapping against a "close" related genome species. The plan is to create a transcriptome with the reads that map (with Trinity) and then map the rest of the reads against this transcriptome. It's still worth to use Bowtie2?

[h.mon]

For mapping RNAseq reads to the "close" reference genome, Bowtie2 is not appropriate.

Mapping the unmapped reads to the assembled Trinity transcriptome with Bowtie2 is fine.

Answer 1

[h.mon]

Yes, you should be using -1 and -2 for paired-end files, and you can't use globing (*.fasta) with Bowtie2. If you want to align several files simultaneously, you have to pass a comma-separated list of files.