Interpreting the meaning of outward facing reads

Question

[jsuzuki]

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.

Answer 1

[jsuzuki]

Deeper into the samtools stats data i found a table showing: for each insert size, how many reads were "innies" vs "outies", and I initially felt like it cleared some things up. The pairs end reads are all about 150 bases in length and almost all the "outie" reads that face away from each other are associated with short insert sizes, less than 150 bases. The average insert length is ~350.

However when i tried to see what that looked like on IGV, i didnt see short outward facing reads. See below.

[jsuzuki]

I looked on IGV and grouped reads by read pair orientation. You can see all the outward facing reads gathered together. Given the chart above, i was expecting short insert sizes, with the two 150base reads overlapping each other, but instead i saw very long insert sizes as pictured below.

Can anyone explain the disconnect between the samtools assessment of outward facing reads (they are all associated with very short insert size) and the IGV representation of outward facing reads, they are associated with all different insert sizes, including very long insert size)? Thank you!!