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markdup error with updated samtools #2117

Description

@kristyhoran

Are you using the latest version of samtools and HTSlib?

Yes
If not, please specify.

Please describe your environment.

  • OS : Linux 4.18.0-553.16.1.el8_10.x86_64
  • machine architecture : x86_64
  • compiler: gcc (GCC) 8.5.0 20210514 (Red Hat 8.5.0-22)

Please specify the steps taken to generate the issue, the command you are running and the relevant output.

I want to run markdup -r. In previous versions,

bwa mem -T 50 -Y -M -R '@RG\tID:Sample\tSM:Sample' -t 8 reference.fa R1.fastq.gz R2.fastq.gz | samclip --max 10 --ref reference.fa.fai | samtools sort -n -l 0 --threads 8 -m 1000M  | samtools fixmate -m - - | samtools sort -l 0  --threads 8 -m 1000M | samtools markdup  -r -s - - > output.bam

Has worked (samtools v1.20). However, when i try to run the same command with version 1.21 the following errorr is reported

samtools markdup: error, no ms score tag. Please run samtools fixmate on file first.

samtools markdup: error, no ms score tag. Please run samtools fixmate on file first.

I note that in the recent release on https://newreleases.io/project/github/samtools/samtools/release/1.21 the following change has been made to fixmate:

samtools fixmate no longer removes the PAIRED flag from reads that have no mate. This is done on the understanding that the PAIRED flag is a sequencing technology indicator not a feature of alignment. This is a change to previous fixmate behaviour. (PR [#2056](https://github.com/samtools/samtools/pull/2056), fixes [#2052](https://github.com/samtools/samtools/issues/2052). Reported by John Wiedenhoeft)

And i wonder if this has caused my problem. I will rollback to 1.20 for now - but thought it would be nice to get your opinion/advice on this.

Many thanks in advance

Cheers
Kristy

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