How to process mutect2 caller after fade out

[deb0612]

Dear sir,
I tried to use docker to process fade on my bam file, and the filtered bam cannot indexed by samtools so that I can't call somatic variants by mutect2.
command I used:
sudo docker run -v pwd:/data blachlylab/fade out -b -c /data/input.hg38.fade.bam >out.hg38.fade.filtered.bam
samtools index out.hg38.fade.filtered.bam
[E::hts_idx_push] Unsorted positions on sequence #1: 203008683 followed by 203008623
[E::sam_index] Read 'A00355:191:HJ2TWDSXY:2:1101:1009:14982' with ref_name='chr1', ref_length=248956422, flags=163, pos=2030d

Even I use samtools sort. The same error keep coming.

[charlesgregory]

You may need to sort your bam again after running fade out. However if using samtools sort doesn't work, you may have something else wrong with your bam file. I need to update our documentation. If using fade out with the -c flag, you should run samtools sort before fade out and after.

[jblachly]

@deb0612 Please let us know if this has fixed your problem -- currently, annotate and out with -c flag commands may "un-sort" your sorted input for reasons of parallelism/speed. We will add warnings in the future.

It is not clear from your comment if you mean using sort AFTER the FADE commands also leads to the same indexing error?

[deb0612]

Dear jblachly,
Unfortunately, the problem is still unsolved.
Whatever I sort my bam file before or after fade out.

[charlesgregory]

@deb0612 We would certainly like to help solve this issue. Could you provide a minimal reproducible example? For example, could you provide SAM line that causes the error when using samtools index?

[deb0612]

Dear @charlesgregory,
Thanks for your reply.
Here is the bam file for tesing
command I used:
$sudo docker run -v pwd:/data blachlylab/fade annotate -b /data/test.bam /data/Homo_sapiens_assembly38.fasta > test.anno.bam
$samtools sort -n test.anno.bam >test.anno.sort.bam
$sudo docker run -v pwd:/data blachlylab/fade out -b -c /data/test.anno.sort.bam > test.anno.sort.filtered.bam
$samtools sort -n test.anno.sort.filtered.bam >test.anno.sort.filtered.sort.bam
$samtools index test.anno.sort.filtered.sort.bam
[E::hts_idx_push] Unsorted positions on sequence #17: 63607429 followed by 63607391
samtools index: failed to create index for "test.anno.sort.filtered.sort.bam"

[charlesgregory]

Try this:

sudo docker run -v pwd:/data blachlylab/fade annotate -b /data/test.bam /data/Homo_sapiens_assembly38.fasta > test.anno.bam
samtools sort -n test.anno.bam >test.anno.sort.bam
sudo docker run -v pwd:/data blachlylab/fade out -b -c /data/test.anno.sort.bam > test.anno.sort.filtered.bam
samtools sort test.anno.sort.filtered.bam >test.anno.sort.filtered.sort.bam
samtools index test.anno.sort.filtered.sort.bam

In order to index the bam you need to do a coordinate sort. samtools sort -n is a queryname sort that is useful for fade out. In order to index the output of fade out you need to re-sort with samtools sort without the -n flag.

[deb0612]

Thanks! It works.