calctruequality in bbmap

[Coyk]

I'm trying to recalibrate Q scores of a NextSeq run using MiSeq contigs assembled with Tadpole. The commands I used to map the reads to the reference were as follows:

>bbmap.sh in=concatABC.fastq.gz outm=mapped.sam ref=./Lpe09_06TdpAssemblies/contigs09_06.fa ignorequality maxindel=100 minratio=0.4 ambig=toss qahist=qahist_raw.txt qhist=qhist_raw.txt mhist=mhist_raw.txt

The command I used to get the calibration matrices was:
>calctruequality.sh in=mapped.sam

I get the following output
java -ea -Xmx57992m -Xms57992m -cp /home/me/bbmap/current/ jgi.CalcTrueQuality in=mapped.sam
Executing jgi.CalcTrueQuality [in=mapped.sam]

Exception in thread "Thread-2" Exception in thread "Thread-1" java.lang.AssertionError: TODO: Encountered a read with 'M' in cigar string but no MD tag and no ScafMap loaded.
at stream.SamLine.toShortMatch(SamLine.java:1212)
at stream.SamLine.toRead (SamLine.java:2015)
at stream.SamLine.toRead (SamLine.java:1875)
at stream.SamReadStreamer$ProcessThread.makeReads (SamReadStreamer.java:206)
at stream.SamReadStreamer$ProcessThread.run (SamReadStreamer.java:135)
java.lang.AssertionError: TODO: Encountered a read with 'M' in cigar string but no MD tag and no ScafMap loaded.
at stream.SamLine.toShortMatch(SamLine.java:1212)
at stream.SamLine.toRead (SamLine.java:2015)
at stream.SamLine.toRead (SamLine.java:1875)
at stream.SamReadStreamer$ProcessThread.makeReads (SamReadStreamer.java:206)
at stream.SamReadStreamer$ProcessThread.run (SamReadStreamer.java:135)
Exception in thread "Thread-3" java.lang.AssertionError: TODO: Encountered a read with 'M' in cigar string but no MD tag and no ScafMap loaded.
at stream.SamLine.toShortMatch(SamLine.java:1212)
at stream.SamLine.toRead (SamLine.java:2015)
at stream.SamLine.toRead (SamLine.java:1875)
at stream.SamReadStreamer$ProcessThread.makeReads (SamReadStreamer.java:206)
at stream.SamReadStreamer$ProcessThread.run (SamReadStreamer.java:135)

The program never quit so I don't know if it had stopped or was hanging but I let it sit there for over an hour. I didn't see /ref/qual/ files being generated. I'm not sure how to proceed. Since the mapped sam file was generated with bbmap using contigs from Tadpole, I'm not sure why there are Ms in the cigar strings. The infile I used for bbmap was made by first concatenating all Read1 from 3 libraries then all Read2 from 3 libraries then those two files concatenated.