Modifying a SAM header after removing all non-primary reads I subset a BAM to only include primary reads using the following samtools commands:

samtools view -F 256 input.bam &gt; input.primaryOnly.sam


Now, in order to convert this SAM into a new subsetted BAM, I would need to add a header. The way I normally do this is with Picard:

java -jar picard.jar ReplaceSamHeader \
      I=input.primaryOnly.sam \
      HEADER=input.header.sam \
      O=new.sam


whereby the header input.header.sam is from the original BAM: 

samtools view -H input.bam &gt; input.header.sam


Then I could convert the SAM into a BAM with 

samtools view -S -b i new.sam &gt; new.bam


My problem is, I'm using the original BAM header. If I look back at the BAM, it now contains wrong information, e.g.

samtools flagstat new.bam


might save that secondary alignments exist, when they don't. 

How do I get a BAM header that makes sense given my new BAM (with only primary alignments)?
Your first command only needs a slight modification to add in -h. This will create a SAM file with a header.

samtools view -h -F 256 input.bam &gt; headered.input.primaryOnly.sam



  -h        Include the header in the output.


If your primary end goal is to create a BAM, then as Pierre has pointed out you can create BAM files directly from samtools view with the -b option:

samtools view -b -F 256 input.bam &gt; input.primaryOnly.bam


And if your end-goal is a sorted BAM file, then pipe the BAM output through samtools sort:

samtools view -b -F 256 input.bam | samtools sort &gt; sorted.input.primaryOnly.bam




So that's fixing what I think your problem is, but what about the specific question that you have asked, i.e. how can a header be added to an existing headerless SAM file?

If you have an existing BAM file, you can use Picard (as you have mentioned), or manually piece the files together with cat, which will have a similar function (although Picard probably includes a few checks to make sure that the header is consistent with the input file):

(samtools view -H input.bam; cat input.primaryOnly.sam) &gt; headered.input.primaryOnly.sam


If the only thing that matters is that the BAM file has correct reference information (which is fairly common), then the -T option can be used with samtools view. Again, -h or -b is required to make sure both the header and the usual SAM fields are displayed:

samtools view -T reference.fa -h input.primaryOnly.sam &gt; headered.input.primaryOnly.sam
samtools view -T reference.fa -b input.primaryOnly.sam &gt; headered.input.primaryOnly.bam