not all reads have the RB tags

[Ding-hang]

I have a question about RB-loss when I want to verify the depth of different RB.
I count the different RB repetition by extract the last column from all the reads, and some reads don't have the RB tags as shown in the picture. I would appreciate it if you could give me some advice about that.
Thank you!

[fa8sanger]

In principle all mapped and properly paired reads should have a RB tag. Have you checked if the RB tag is not in the last column? Feel free to send a mini version of the bam and I’ll have a look. On 20 Dec 2022, at 14:36, Ding-hang ***@***.******@***.***>> wrote: [飞书20221220-221545] [user-images.githubusercontent.com]<https://urldefense.proofpoint.com/v2/url?u=https-3A__user-2Dimages.githubusercontent.com_108725789_208687516-2D6b6f3565-2D10cc-2D4250-2Dba7a-2D0b9894c72db8.jpg&d=DwMFaQ&c=D7ByGjS34AllFgecYw0iC6Zq7qlm8uclZFI0SqQnqBo&r=v9-R7fUmjpv-9Zaqyk1nlnlOC3qPkTEJz5tyYxg2uec&m=EecB5fkqPiCH8p21iKJtfYYF-L-po-ZrmmPWlHloR1oocpfC3eR7sPZhAlzo7K01&s=SAGB_Mdwb93-4x7yFCuHc1-UQqV_T12ckU6dxZKN3kA&e=> I have a question about RB-loss when I want to verify the depth of different RB. I count the different RB repetition by extract the last column from all the reads, and some reads don't have the RB tags as shown in the picture. I would appreciate it if you could give me some advice about that. Thank you! — Reply to this email directly, view it on GitHub [github.com]<https://urldefense.proofpoint.com/v2/url?u=https-3A__github.com_cancerit_NanoSeq_issues_56&d=DwMFaQ&c=D7ByGjS34AllFgecYw0iC6Zq7qlm8uclZFI0SqQnqBo&r=v9-R7fUmjpv-9Zaqyk1nlnlOC3qPkTEJz5tyYxg2uec&m=EecB5fkqPiCH8p21iKJtfYYF-L-po-ZrmmPWlHloR1oocpfC3eR7sPZhAlzo7K01&s=M9iA3zvoV-SRbzC4xzv6cFvA2e7HMwkcTGMLbNMXaM0&e=>, or unsubscribe [github.com]<https://urldefense.proofpoint.com/v2/url?u=https-3A__github.com_notifications_unsubscribe-2Dauth_ADNUT3PPZ5RNKUGLPJEUPFTWOG753ANCNFSM6AAAAAATESJRQQ&d=DwMFaQ&c=D7ByGjS34AllFgecYw0iC6Zq7qlm8uclZFI0SqQnqBo&r=v9-R7fUmjpv-9Zaqyk1nlnlOC3qPkTEJz5tyYxg2uec&m=EecB5fkqPiCH8p21iKJtfYYF-L-po-ZrmmPWlHloR1oocpfC3eR7sPZhAlzo7K01&s=WEnvFrJMf_kpqrTDnsT4LMVYNNm-wzXAzvdQ1CMtAss&e=>. You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

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[Ding-hang]

Following the workflow in the github, I get this file from the original document.
And I have checked the RB tags as the picture show. The RB tags are all in the last column without other operation on this bam file.

[fa8sanger]

All good then, right?

[Ding-hang]

Thanks for your reply!
I‘m sorry about I foget pointing out there are 66053 (6001205-5935152) reads have no RB tag.
The sum of the these reads without RB tag is fit the loss(16358+15821+17319+16555=66053).
I wonder if this result fit the expectation, and I filter these reads without RB tag in following process, which makes me worry.

[fa8sanger]

In principle all reads should have a RB tag after running bamaddreadbundle. Recently we developed a version that also saves reads without RB but I don't think you run that one. I have no idea what happened in your case with those reads. I would need to check your bam file.

[fa8sanger]

Also, could you try this: samtools view your_bam.bam | grep -v "RB:" ?

[Ding-hang]

The bam file turely have some RB tags loss.

The number of the reads fits the number I given.

I guess it's something wrong in my bamaddreadbundle, but I have no idea about that.