I attached here the workflow that for a while we used to analyze WGS data and process fastq files to arrive to variant calling and annotation. This workflow does not work anymore, and it seems it depends on unified genotyper being deprecated.
Compared to the workflow represented here I already substituted "BWA for illumina " with "BWA (<100bp). Since the output of the new BWA tool is already BAM i also removed the SAM filter and SAM to BAM conversion. I added instead “Mark Duplicates” and left in place “add or replace groups”.
After this point i tried uncountable combination of variant calling and annotation tools with very poor results. The final tabular files i managed to obtained contained either few hundreds (too little) variants or 10 or > thousands (too many), but in no case they would include the background mutation typical of the parental strain that i mutagenized. I can see the mutation when looking at the alligned sequence in the UCSD browser. So i deduced that there was something wrong in the tool i used or the parameter i imposed.
In light of these considerations, I’d like to ask the following:
- the step i took so far to modify the old workflow are correct? is there something i should add or remove?
- Which variant calling tool is the most suitable to detect germline mutations ( i work with C.elegans) and which follow up annotation and filtering tools should i use?
From what i read the best new alternative to Unified genotyper is Haplotype Caller but it is not available among the Galaxy tools.
Any advice or input will be very welcome! Thanks a lot!