Error in Generating sorted.bam

Question

[jaqx008]

Hi all, I posted a question few weeks back and I haven't been able to resolve the issue with my .Bam and sorted.bam files.

I ran the following command and for some reason, I can't generate my required files which I need for running the GATK command. (my reference genome is an index file). also, I'm a Beginner.

$ bowtie2 -x /Volumes/500GB/pavelAssemblyBowtieIndex -1/Volumes/500GB/Illumina_Run_814/Run_814/Project_klimov/Sample_28017/28017_GTCCGC_L006_R1_001.fastq -2 /Volumes/500GB/Illumina_Run_814/Run_814/Project_klimov/Sample_28017/28017_GTCCGC_L006_R2_001.fastq -S dustMapping.sam && samtools view -S -b dustMapping.sam -o dustMapping.bam && samtools sort dustMapping.bam -odustMapping.sorted.bam && samtools index dustMapping.sorted.bam -o dustMapping.sorted.bam.bai && rm dustmapping.sam && dustmapping.bam
(ERR): "/Volumes/500GB/pavelAssemblyBowtieIndex" does not exist or is not a Bowtie 2 index
Exiting now ...

[jared.andrews07]

Are all of your index files prefixed with 'pavelAssemblyBowtieIndex' and in the /Volumes/500GB/ folder? Cause that's what it's looking for - /Volumes/500GB/pavelAssemblyBowtieIndex.1.bt2, etc, and it can't find them.

Also, the -o argument isn't needed for samtools index, it'll create the new file with the extension as you have it without the argument.

[jaqx008]

All the bt2 are in the pavel folder. I would have used the reference genome but I only have the indexes.

[ori]

Could you get the dustMapping.sam that is the first output file?
If not, please check whether there are bowtie"2" index files near the reference file (pavelAssemblyBowtieIndex .fa).

Bowtie2 index : .1.bt2, .2.bt2, .3.bt2, .4.bt2, .rev.1.bt2, and .rev.2.bt2.
Bowtie 2's .bt2 index format is different from Bowtie 1's .ebwt format, and they are not compatible with each other.
http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml

[jaqx008]

Ok let me go ahead and run only the sam output. But there is no .fa file. I only have the indexes.

Answer 1

[swbarnes2]

Read what the error message is. Something is wrong with your path to the index. Are you 100% sure it's there, and that you have the right path? Are you sure it was made with bowtie2, and not bowtie?

[jaqx008]

I didn't actually make the index files. I'm not sure if they used bowtie2 or bowtie

[jaqx008]

I saw an error in the path and the command is running now. I used the same command and I hope it doesn't generate error as before. Thanks