Interpreting the meaning of outward facing reads

Interpreting the meaning of outward facing reads

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Sorry i have lots of data and im not sure how to interpret.

I took mRNA created by IVT from our plasmid and performed paired end Illumina sequencing.
I used samtools stats to get info about the various samples: read length ~150, insert size ~350, MapQ ~35
Most reads are inward facing FR pairs, but surprisingly (to me) there are also outward facing RF pairs.
I am looking at the percentage of outward facing pairs and wondering if these are evidence of the plasmid being compromised by some level of rearrangements or if its just normal sequencing messiness.

What is the expected rate of “outie” reads if the plasmid is all still perfect?
At what point should i be concerned?
1% 5% 10%?
Thank you.


Illumina


sequencing

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