Empty Counts File

[sanchari24]

Hello, I am following the pipeline of finding alternative splicing analysis using QoRTs and JunctionSeq.
I am working on tomato rna-seq data
I have used HISAT2 for alignment and used "--minMAPQ 60".
QoRTs ran without errors but the two count files generated are empty. I tried to get the counts only for known splice sites.
I have attached the summary, log and gtf file for your reference. Any lead from your side will be very helpful.

ITAG3.2_genomic.gtf.gz
QC.summary.txt
QC.v1id5eVrCLb8.log

[hartleys]

The QC command requires a GTF file, NOT the flat GFF file.

…

On Wed, Aug 1, 2018, 8:31 AM sanchari24 ***@***.***> wrote: Hello, I am following the pipeline of finding alternative splicing analysis using QoRTs and JunctionSeq. I am working on tomato rna-seq data I have used HISAT2 for alignment and used "--minMAPQ 60". QoRTs ran without errors but the two count files generated are empty. I tried to get the counts only for known splice sites. I have attached the summary, log and gtf file for your reference. Any lead from your side will be very helpful. ITAG3.2_genomic.gtf.gz <https://github.com/hartleys/QoRTs/files/2249404/ITAG3.2_genomic.gtf.gz> QC.summary.txt <https://github.com/hartleys/QoRTs/files/2249399/QC.summary.txt> QC.v1id5eVrCLb8.log <https://github.com/hartleys/QoRTs/files/2249400/QC.v1id5eVrCLb8.log> — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#69>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ACwu7Bm7DCv9nQwqMs5Y2tFtT-hw1_kNks5uMZ-ogaJpZM4VqaLL> .

[sanchari24]

I gave the genomic GTF file attached above as the input, with the commands:

java -jar QoRTs-STABLE.jar QC
--stranded
--minMAPQ 60
--runFunctions writeKnownSplices,writeNovelSplices,writeSpliceExon
/home/workstation3/rtgr_rna-seq/bam_files/TFRR_1.sorted.bam
/home/workstation3/rtgr_rna-seq/input/ITAG3.2_genomic.gtf.gz
/home/workstation3/rtgr_rna-seq/bam_files/TFRR_1_count \

The "QC.spliceJunctionAndExonCounts.forJunctionSeq.txt.gz" is empty. and I am getting this error:
"Error message: "IMMPOSSIBLE STATE! FATAL ERROR! qcJunctionCounts.writeOutput, writing forSpliceSeq"
Thanks for your help.

QC.iM2kH5BvR60T.log

[hartleys]

Hrmm. My suspicion is that there are genes in the GTF with unexpected characters. In particular, colons in gene names would cause this error to occur.

…

On Thu, Aug 2, 2018, 2:26 AM sanchari24 ***@***.***> wrote: I gave the genomic GTF file attached above as the input, with the commands: java -jar QoRTs-STABLE.jar QC --stranded --minMAPQ 60 --runFunctions writeKnownSplices,writeNovelSplices,writeSpliceExon /home/workstation3/rtgr_rna-seq/bam_files/TFRR_1.sorted.bam /home/workstation3/rtgr_rna-seq/input/ITAG3.2_genomic.gtf.gz /home/workstation3/rtgr_rna-seq/bam_files/TFRR_1_count \ The "QC.spliceJunctionAndExonCounts.forJunctionSeq.txt.gz" is empty. and I am getting this error: "Error message: "IMMPOSSIBLE STATE! FATAL ERROR! qcJunctionCounts.writeOutput, writing forSpliceSeq" QC.iM2kH5BvR60T.log <https://github.com/hartleys/QoRTs/files/2252331/QC.iM2kH5BvR60T.log> — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#69 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ACwu7Mi7x-bi-xAlp_wed42-3KlUJkkoks5uMpuqgaJpZM4VqaLL> .

[sanchari24]

I see, that's not a good news for me. I will scan the gtf file properly and will let you know.
Thanks a lot.

[sanchari24]

Hello, I went back to the older genome version so that I can use the Ensembl gtf file for my organism. QoRTs ran without any errors. However, when I ran JunctionSeq in R, I got the following error:
Error in DESeqDataSet(se, design = design, ignoreRank) :>
all samples have 0 counts for all genes. check the counting script.

I then checked the file "QC.spliceJunctionAndExonCounts.forJunctionSeq". It had zero counts for all the entries in all the samples. I am unable to understand the reason for this and will be very greatful if you could look into my files.
Thanking You
QC.summary.txt
QC.spliceJunctionAndExonCounts.forJunctionSeq.txt.gz
QC.spliceJunctionCounts.knownSplices.txt.gz
QC.spliceJunctionCounts.novelSplices.txt.gz

[hartleys]

There are a lot of different things that could have gone wrong.

• Do the GTF and the BAM file use the same chromosome names? (ie: "chr1" vs just "1")
• Is the ensembl GTF built for the same genome version you are using?

Can you attach the log?

[gk7279]

Hi there-

my genomes have underscores and no colons or special characters. but still it terminates with the same error as the original poster. my chromosome names are all matching. my log file is attached. can you please help?
qc.log

[hartleys]

Hmm. Very strange. Could I see the GTF file?

Also, try running this:

java [Java Options] -jar QoRTs.jar makeFlatGff --stranded ip.gtf ip.flat.GFF

And maybe post an excerpt from the GFF file?