read groups

[sasignor]

This is a problem for which there are many posts already. Yet having spent two days googling my problems, and having them unresolved, I am going to ask. I have 17 samples from two subspecies that I want to eventually analyze in vcftools for some basic population genetics. From what I can tell, this requires them to be all in one file with some sort of distinguishing header for the different subspecies. So I am trying to add headers to the .sam files and then merge them such that this will be possible.

I have tried using sampe:

./bwa sampe -r "@RG\tID:3\tSM:3\tPL:illumina" reference.txt in.sai in.sai in.fastq in.fastq > header.sam

And this does add the header, but just as one line in the file @RG. Adding it using samtools merge -rh and a text file does not seem to work at all.

Using picard to merge the files seems to maintain the headers, again as one line.

java -Xmx2g -jar MergeSamFiles.jar

but when it is finalized as a vcf all distinguishing tags are gone.

Perhaps I am mistaken as to the input that vcf requires? How else is it supposed to tell between populations?

Are these headers not enough to tag the reads?

[dpryan]

If you already have the alignments, just use picard tools AddOrReplaceReadGroups (adding read groups should alter every alignment, so if you "samtools view sample1.bam | head" before and after adding read groups you should see a difference). We'd have to see and example of from the VCF file to see if things are going amiss there.

[rskr]

This is a problem for which there are many posts already. Yet having spent two days googling my problems, and having them unresolved, I am going to ask. I have 17 samples from two subspecies that I want to eventually analyze in vcftools for some basic population genetics. From what I can tell, this requires them to be all in one file with some sort of distinguishing header for the different subspecies. So I am trying to add headers to the .sam files and then merge them such that this will be possible.

I have tried using sampe:

./bwa sampe -r "@RG\tID:3\tSM:3\tPL:illumina" reference.txt in.sai in.sai in.fastq in.fastq > header.sam

And this does add the header, but just as one line in the file @RG. Adding it using samtools merge -rh and a text file does not seem to work at all.

Using picard to merge the files seems to maintain the headers, again as one line.

java -Xmx2g -jar MergeSamFiles.jar

but when it is finalized as a vcf all distinguishing tags are gone.

Perhaps I am mistaken as to the input that vcf requires? How else is it supposed to tell between populations?

Are these headers not enough to tag the reads?

I use the -h option in samtools merge to specify a separate header file, for the header file I print the header from one of the bam file then concatenate the @RG tags for all of the samples to the header.

Something like:

Samtools view -h file1.bam | grep ^@ > header.txt
Cat rg.txt header.txt > rg_header.txt
Samtools merge -h rg_header.txt out.bam file*.bam

Where rg.txt has the @RG tags.

It is a weird operation IMO, if there were a simpler way to do it, I would like to know, I can post more details if it isn't clear how to do it this way.