Hello,

I am trying to generate a .vcf file and call variants in RNA-Seq data, as per the GATK3 tutorial: https://software.broadinstitute.org/gatk/documentation/article.php?id=3891

I can get all the way through making a filtered .vcf file, but the resulting file has no entries - i.e., the .vcf file is only 47 lines, and all of that is header information. The columns are empty. The unfiltered .vcf file in the HaplotypeCaller is also empty.

My questions are:

- First - as a sanity check - this is an unlikely outcome, right? It's not that my RNA-Seq coverage was too shallow (~30,000,000 paired end reads) and therefore we are unable to detect any variants?
- Second - if we should, indeed, see some variants in that file - since it's hard to tell if there are variants at all until you actually generate a .vcf file (i.e., you can't look in a bam file and decide that variants exist) - what parameters should I tweak in order to try and get variants in my final .vcf file? I'm not sure what intermediate steps I can look at to troubleshoot where in the pipeline things are going wrong.

I didn't perform Indel Realignment (step #4) or base recalibration (step #5) because it was unclear to me from the tutorial if this was necessary for RNA-Seq. Right before step 6, I also had to use ReorderSam and BuildBamIndex in order to make the intermediate bam files generated match the ordering of the .fa file I am using as an index. I'll work on getting base recalibration working next, but if anyone has additional suggestions or answers to the above, I'd appreciate it.

Thank you for your help!

Kristin
Thank you everyone for providing valuable feedback that helped me narrow down the world of what could possibly be going wrong and identifying the key issue.

Ultimately, the problem was that I hadn't noticed that at some point the second alignment had failed, and I was trying to run all these commands on an empty .bam file. Thus - the problem wasn't the .dict at all - but the files that were being used for calling.

I'm a little confused about how I missed this because I had been using samtools to take a peek at every file and make sure they looked fine - but this troubleshooting process has taken so many iterations that I must have missed something.

I was inspired by this post, where someone encounters a similar issue: https://gatkforums.broadinstitute.org/gatk/discussion/10130/gatk-3-8-dictionary-cannot-have-size-zero 

Now that I went back and make sure the first and second alignment steps produced .bam files, I do not get the .dict error, and my .vcf files have variants written in them.

Thank you all!