Question: SAM flags for strand-specific protocols
1
gravatar for Pfs
3.8 years ago by
Pfs490
United States
Pfs490 wrote:

I would like to understand how to interpret the SAM flags (relatively to strandness) for both strand-specific protocols and non-strand-specific protocols. Are the following making sense?

 

5'--------------------------------------------3'

read/1 -------->  <--------- read/2

/1 has flag 99, /2 has flag 147

 

read/2 -->    <--- read/1

/2 has flag 163, /1 has flag 83

 

How does this change when the protocol is strand-specific?

 

Thanks!

 

rna-seq sam • 1.6k views
ADD COMMENTlink modified 3.8 years ago by Asaf5.5k • written 3.8 years ago by Pfs490
1
gravatar for Asaf
3.8 years ago by
Asaf5.5k
Israel
Asaf5.5k wrote:

If I understood you correctly, this won't change when the experiment is strand specific. In the current situation you get mappings to the positive and reverse strands but the strand is meaningless, when the experiment is strand specific you'll also get mapping to the positive and reverse strands but they will be meaningful. 

ADD COMMENTlink written 3.8 years ago by Asaf5.5k
1
Its simply, when you turn RNA to DNA as part of the RNA-seq protocol, you lose the information about whether the RNA transcript came from the forward or reverse strand. So what you sequence in the sequencer could tell you where the RNA came from, but not if its AAATC or if its actually TTTAG and you just happened to have sequenced the other side of the cDNA. Newer protocols chemically tag the second DNA strand synthesize by using dUracil unstead of Thymine. Then right before PCR, all the strands with the Uracil are degraded enzymatically, so the result is you know not only where the RNA was made, but also which strand, forward or reverse. The BAM file doesnt know or care about any of this :) Its up to you, if you used a strand-specific protocol, to do something meaningful with the read sequence and the flag information :)
ADD REPLYlink written 3.8 years ago by John12k
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