featureCounts, GZIP ERROR:-2

Question

[bisht20diksha]

Hello. I am new to differential gene expression analysis. I was trying the tutorial of griffith test data provided in BIOSTAR HANDBOOK. I have done the alignment using HISAT2 and when I am doing quantification using the

featureCounts

I am getting the GZIP ERROR:-2 as follows:

GZIP ERROR:-2

the data is paired ended so I am using the same command given in the manual of subhead package.

featureCounts -p -a annotation.gtf -t exon -g gene id -o counts.txt mapping results PE.bam

Also, the path and working directory all are fine, still, I don't know why this error is coming up. Please suggest as I am stuck at this step and not able to proceed.

Thanks in advance!

[GenoMax]

That is odd. There should be no GZIP involved in running featureCounts. Can you post your exact command?

[bisht20diksha]

I am also encountering this error for the first time. yes, the command is used is:

/Users/gkp8/src/subread-1.5.1-source/bin/featureCounts -p -t exon -g gene_id -a refs/ERCC9.2.gtf -o counts.txt bam/HBR_1.bam bam/HBR_2.bam bam/HBR_3.bam

[GenoMax]

As indicated by @Shalu your BAM files may have some problems. Did you get any errors in prior steps where you were generating these files? Have you looked through the logs? What do you see if you try the command file bam/HBR_1.bam (and other BAM files above)?

[bisht20diksha]

No, I did not find any error while creating bam files.

I have generated a directory named griffith and then I have downloaded the data from:

This is the URL for the data

URL=http://data.biostarhandbook.com/rnaseq/projects/griffith/griffith-data.tar.gz

Download and unpack the data

curl $URL | tar zxv

and then I created bam directory within griffith and aligned reads using:

hisat2 $IDX -1 $R1 -2 $R2 | samtools sort > $BAM

Okay! Sir/Ma'am I will look for bam files validity.

[bisht20diksha]

when i tried file bam/HBR_1.bam , I am getting this error:

bam/HBR_1.bam: gzip compressed data, extra field

is it because these files are zipped one? what I have to do next?

[GenoMax]

That is what you should see with normal BAM files. So this part should be ok.

Not sure why you posted below that you are getting an error with hisat2. Are you trying your own data files now?

[bisht20diksha]

no, I am trying with the same files, I did not check or hisat2 earlier. I just checked for BAM and it was there in new bam directory. I didn't have much experience with iMac.

So, now, In my Linux system, I have installed HISAT2 using conda and then I ran hisat2 to check whether its working or not. I am getting this error at the last line after the usage:

(ERR): hisat2-align exited with value 1

and with the same hisat2, when I am trying to build index using

hisat2-build $REF $IDX

I am getting a similar error and bam files of 0 bytes.

(ERR): hisat2-align exited with value 1

I think the error is because of indexing issues (with HISAT2).

[GenoMax]

If you feel that you made a mistake somewhere upstream in the workflow then go back and check all previous files starting at the top with raw data. If you used conda to install hisat2 then there should not be much of an issue.

[bisht20diksha]

okay I will check from the first step again.

Answer 1

[Shalu Jhanwar]

It seems the error encounter due to BAM file input with some bad blocks. Did you check if the validity of the BAM file that is generated correctly and not damaged/corrupted? I'd suggest trying a couple of things:

1. Could you try re-running the command with SAM file as input in featureCounts?

2. Could you try running the command on a different BAM file (maybe from publically available data?)

Have a look at a discussion forum regarding similar error: https://groups.google.com/forum/#!topic/subread/S4smWRfBNPM

Hope this helps!

[bisht20diksha]

No, I did not check the validity as I was following the instructions provided in the tutorial. Corrupted BAM could be the problem.

I will try these points suggested by you and will update then.

thanks for the suggestions.

[bisht20diksha]

Hello Ma'am @Shalu I checked my all commands again and tried it on my Linux system also.

I found that when I am trying to align using HISAT2, I am getting the following error:

(ERR): hisat2-align exited with value 1

because of which, I am getting empty BAM files (0 bytes). Please help with this, I am unable to figure this out!

Thanks in advance