I'm trying out different variant calling software on non-matched somatic samples, where the pcr has been performed using an Agilent kit called Haloplex HS.
At the moment I'm trying VarDict, and I want to try to run VarDict in amplicon-aware mode. The problem is, amplicon-aware mode requires a bed file with start and end positions both with primers and without primers, and Haloplex HS doesn't use standard pcr primers. As can be seen in this image, Haloplex has a molecular barcodes and indexes in addition to a generic pcr primer.
Is it possible to run amplicon-aware calling on barcoded-data? I've tried adding two columns to my amplicons bed file which are the original amplicon start/end points moved one base back/forward, but I get the following error:
Exception in thread "main" htsjdk.samtools.SAMException: Malformed query; start point 139389396 lies after end point 138394717 at htsjdk.samtools.reference.IndexedFastaSequenceFile.getSubsequenceAt(IndexedFastaSequenceFile.java:215) at com.astrazeneca.vardict.ReferenceResource.retriveSubSeq(ReferenceResource.java:31) at com.astrazeneca.vardict.ReferenceResource.getREF(ReferenceResource.java:50) at com.astrazeneca.vardict.VarDict.ampVardictNotParallel(VarDict.java:5577) at com.astrazeneca.vardict.VarDict.start(VarDict.java:69) at com.astrazeneca.vardict.Main.run(Main.java:138) at com.astrazeneca.vardict.Main.main(Main.java:25)
Does anyone have any ideas on how to approach this?