Mark -
Just guessing: One may need to specify "merge -f" in case an
earlier invocation of the command has created an empty file with
the path generated by "jp(bamFolder, sample + ".bam")". Also may
need to specify "merge -n" if, in fact, the output from bwa is
already properly name-sorted. Again, just guessing.
- tom blackwell -
On Fri, 12 May 2017, Margres, Mark J wrote:
> Hello,
>
> I am having an issue with merging different bam assemblies using samtools.
> In my pipeline, I merge reads using flash2, and then perform reference-based
> assemblies using bwa, one assembly uses just the merged reads and a second
> assembly uses just the unmerged reads. I then attempt to merge the
> assemblies using samtools and get the following output:
>
>
> [W::bam_merge_core2] No @HD tag found.
>
> I am assuming this is due to the unmerged read assembly having a different
> header than the merged reads, but I am not sure why (and also not sure if
> this is a bwa issue or a samtools issue). Is there a way for me to merge and
> then sort the assemblies with maintaining the correct header tags? I have
> the original assemblies to work with, but have assembled abut a dozen genomes
> and would prefer to not re-assemble if possible. Code is below. Thanks.
>
> # Run BWA to map PE samples to reference genome
> # -t number of threads -R read group header
> logFile = jp(resultsDir, sample + '_mapping.log')
> cmd = ' '.join(["/home/mark.margres/bwa-0.7.15/bwa mem -t 50 -R
> '@RG\tID:bwa\tSM:" + sample + "\tPL:ILLUMINA'",
> bwaIndex, jp(resultsDir, sample + "_cleaned_PE1.fastq.gz"),
> jp(resultsDir, sample + "_cleaned_PE2.fastq.gz"), "|
> samtools view -bS -@ 50 -o", jp(bamFolder, sample + "PE.bam"),
> "2>", logFile])
> log(cmd, logCommands)
> os.system(cmd)
> # Run BWA to map SE samples to reference genome
> cmd = ' '.join(["/home/mark.margres/bwa-0.7.15/bwa mem -t 50 -R
> '@RG\tID:bwa\tSM:" + sample + "\tPL:ILLUMINA'",
> bwaIndex, jp(resultsDir, sample + "_cleaned_SE.fastq.gz"),
> "| samtools view -bS -@ 50 -o", jp(bamFolder, sample + "SE.bam"),
> "2>>", logFile])
> log(cmd, logCommands)
> os.system(cmd)
> # merge and sort
> cmd = ' '.join(['samtools merge -c', jp(bamFolder, sample + ".bam"),
> jp(bamFolder, sample + "PE.bam"), jp(bamFolder, sample + "SE.bam")])
> log(cmd, logCommands)
> os.system(cmd)
> # sort bam file; -@ number of threads
> cmd = ' '.join(['samtools sort -o', jp(bamFolder, sample) + "sorted.bam",
> ' -@ 50', jp(bamFolder, sample + ".bam")])
> log(cmd, logCommands)
> os.system(cmd)
> # Mark and remove PCR duplicates
> cmd = ' '.join([picard + "MarkDuplicates INPUT=" + jp(bamFolder, sample +
> "sorted.bam"), ' OUTPUT=' + jp(bamFolder, sample + "_markdup.bam"),
> ' METRICS_FILE=' + jp(bamFolder, sample + ".metrics"), '
> REMOVE_DUPLICATES=true ',
> ' ASSUME_SORTED=true VALIDATION_STRINGENCY=LENIENT', '>>',
> logFile, '2>&1'])
> log(cmd, logCommands)
> os.system(cmd)
> # Index bam file:
> cmd = ' '.join(['samtools index', jp(bamFolder, sample) + "_markdup.bam"])
>
> ----------------------
> Mark J. Margres, Ph.D.
> Postdoc, Storfer Lab
> School of Biological Sciences
> Washington State University
> Pullman, WA 99164-4236
>
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