editing header files

[sasignor]

I will admit I have little understanding of exactly how read groups work with regards to tags within the file instead of just the header. Here is my problem:

1. I originally processed all my files with @RG tags for ID and SM, where ID was always 1 (the problem) and SM was the sample name (which is fine).

2. A downstream application I use has problems with the ID:1 tag, and I need it to be the sample name (I think, the downstream application has little documentation, but on other files where I did this originally it worked).

4. Using samtools view -H file > file.txt I edited each header file to have ID: sample id then used samtools reheader.

3. Before using the downstream application, I need to merge my files and I did this:

/home/jdk1.7.0_55/bin/java -Xmx2g -jar ../bin/picard-tools-1/picard-tools-1.89/MergeSamFiles.jar

followed by a list of I= and O=

4. The error:
Exception in thread "main" net.sf.samtools.SAMFormatException: SAM validation error: ERROR: Record 1, Read name M01533:70:000000000-A5W2F:1:2113:28047:12326, RG ID on SAMRecord not found in header: 1
at net.sf.samtools.SAMUtils.processValidationErrors(SAMUtils.java:448)
at net.sf.samtools.BAMFileReader$BAMFileIterator.advance(BAMFileReader.java:541)
at net.sf.samtools.BAMFileReader$BAMFileIterator.<init>(BAMFileReader.java:500)
at net.sf.samtools.BAMFileReader$BAMFileIterator.<init>(BAMFileReader.java:488)
at net.sf.samtools.BAMFileReader.getIterator(BAMFileReader.java:290)
at net.sf.samtools.SAMFileReader.iterator(SAMFileReader.java:322)
at net.sf.picard.sam.MergingSamRecordIterator.startIterationIfRequired(MergingSamRecordIterator.java:100)
at net.sf.picard.sam.MergingSamRecordIterator.hasNext(MergingSamRecordIterator.java:115)
at net.sf.picard.sam.MergeSamFiles.doWork(MergeSamFiles.java:147)
at net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:177)
at net.sf.picard.sam.MergeSamFiles.main(MergeSamFiles.java:79)


So, can you not edit @RG tags after the file has been created? The very existence of samtools reheader seems to support that you can.....Is there a way of doing this that does a better job, and actually changes the read tags, assuming some kind of tag on each read is the problem?

[dpryan]

Just use picard tools AddOrReplaceReadGroups. That'll make your life easier.

Unlike many other things, the read group ID is stored as text in each alignment, so changing the header alone won't be sufficient. I agree that this is somewhat annoying.

[sasignor]

Yes that seems to work. The existence of samtools reheader seems to be somewhat pointless.

I still wish I knew more about how each tag is attached to each read, and how that is then interpreted down the line, but oh well.

[dpryan]

It's fine if you just want to change chromosome names (e.g., from UCSC to Ensembl names).

Regarding understanding how things are stored, this is partly covered in the SAM spec. in the section about BAM format, but it's generally easier to remember if you've played with the samtools source code a bit.