Reads have rejected SAM flags

[sinhaeti]

Hi!

I'm having trouble with the output of the script smmipcollasper.py in tools. A large portion of my reads have rejected SAM flags. What flags are causing this? How can I avoid filtering via SAM flags?

Prior to running the script, basic filtering was already completed. For example (in the picture below), I started with 7450 reads. However, over 3000 filtered via SAM Flags.

[augustboyle]

Hello, The SAM flag check makes sure that the reads are properly paired and have the correction orientation. If the SAM flags do not show properly paired reads, then that usually means something is wrong with the data or it isn’t being aligned properly. You should try looking at the flags and discerning why they aren’t ‘perfect.’ https://broadinstitute.github.io/picard/explain-flags.html Evan On January 22, 2019 at 3:13:25 PM, sinhaeti ([email protected]) wrote: Hi! I'm having trouble with the output of the script smmipcollasper.py in tools. A large portion of my reads have rejected SAM flags. What flags are causing this? How can I avoid filtering via SAM flags? Prior to running the script, basic filtering was already completed. For example (in the picture below), I started with 7450 reads. However, over 3000 filtered via SAM Flags. [image: image] <https://user-images.githubusercontent.com/16585341/51571705-d8cfdd00-1e57-11e9-8409-0a7de3f6055d.png> — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#38>, or mute the thread <https://github.com/notifications/unsubscribe-auth/AF01p2ZbUBjbJCtaW_Dhc4wmi3bJxkM8ks5vF5sVgaJpZM4aNtd9> .

[sinhaeti]

I already checked for the SAM flags. The flags I found were not concerning (83, 163, 2131). Is there a way to avoid filtering via SAM flags?

Prior data filtering already removed reads that were: too long/short, not paired, etc.

[augustboyle]

It seems likely that the reads are being rejected because of a supplementary alignment (flag 2131), see this page: https://www.biostars.org/p/308853/

If you want to include those reads then add 2131, 2195, 2211, and 2147 to the good flags frozenset in the genome_sam_collapser.pyx on line 669.

However, given the way the MIP recognition works, it seems likely that it will fail MIP recognition. In that case, the MIP arms would not be trimmed. You can try a newer MIP analysis pipeline that may have more advanced QC: https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1007956