Error : .bam is not properly preprocessed.

[zz311784]

Hi,
I keep encountering this error saying: bam is not properly preprocessed while running dsa. Here is my bam: LH00373:276:227J7NLT1:2:1101:27962:1192 83 1 16717878 60 144M = 16717664 -358 TGTGGCCTGGGGCAGCTCAAGCCTCTGCCCTACCCAGCTCCCAGCCTTCATATCCACAGCCTCGGTGAGCAGGAATCCCTACCCCCAAGATAGGAGGTTAAGTGGGTACCCCAGCCCCCAACCCCAATTCCCAGCTGCTCTTCC I-IIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIII NM:i:1 MD:Z:84G59 AS:i:139 XS:i:108 rb:Z:CCT mb:Z:TGT MQ:i:60 ms:i:5744 mc:i:16717664 MC:Z:144M rc:i:16718021

Can you help me out?

Thanks!

[fa8sanger]

It seems you haven't run bamaddreadbundle first. It will create a RB tag combining rc, mc, mb and rb. Check the readme on how to preprocess the bam file in case you are missing any of the other steps

[zz311784]

Thanks for the help! I regenerated the bam file:LH00373:276:227J7NLT1:2:1101:49311:1144 83 17 21598579 60 144M = 21598490 -233 TACTAATGCCCACCCCTCCCTCTACTGGACACAGTCAGCTCTAGGAAGGGGAGAGAAAATCCATCTAGAATGAGGGCAACTGCTGTGGCTGGGGAAAGATGCGACAGGGCAGATGAGCTTGTCCTGGTGCCCATCTCAGGCCCC IIIIIII-9IIIIIIIIIII9IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII9IIIIIIIIII NM:i:0 MD:Z:144 AS:i:144 XS:i:24 RB:Z:17,21598490,21598722,CTG,TAA but the error was not resolved.

[zhangzhen]

@zz311784 you can replace
NLINES=samtools view $cram | head -1 | grep rb: | grep rc: | grep mb: | grep mc: | wc -l || true
with
NLINES=samtools view -F 2820 $cram | head -1 | grep rb: | grep rc: | grep mb: | grep mc: | wc -l || true
in process NANOSEQ_ADD_RB in modules/NanoSeq_aux.nf.
This change fixes the same error i encountered. I hope it can fix your error too.

[fa8sanger]

Thank you. I thought we had already fixed that problem. I cannot alter the main repo, but I have a forked branch here: https://github.com/fa8sanger/NanoSeq/tree/develop
Feel free to use that one

[zz311784]

Thank you all for the instructions! I was running manual executions. So, should I make the corresponding changes in bamaddreadbundles.cc or somewhere else?

[fa8sanger]

I already made those changes in NanoSeq_aux.nf, no need to change bamaddreadbundles.cc