Hi all,

I have read through many posts about insert size here. And see [a very good answer about the insert size][1]. 

It is still not so clear for me to understand insert size. I hope some experts can make it clearer.

As illustrated in [a good blog][2] and [a good anwser][3], the "insert size"=sequence between adapters (actually encompasses R1 and R2 as well as the unknown gap between them) and it is also known that [the ninth column of the SAM file (TLEN) represents the insert size][4]

However, here are some things I still don't understand.

First, in RNA seq data, if the alignments are spliced, and the TLEN reports the distance from the 5'-most to 3'-most position ([if my understanding is right][5]).  So according to my understaning the TLEN number will include the possible introns  which means the TLEN would be unsally longer than "actual insert size"? 

Second, if we are mapping DNA sequences, then the fragment length and "insert size"/"template length" are the same?

Third, how Picard tools [CollectInsertSizeMetrics ][6] actually do to calculate the insert size distribution of a paired-end library, does it only use the TLEN or exclude possible introns?

Any answer to help me better ubderstand this conception will be greatly appreciated.


  [1]: https://www.biostars.org/p/94246/
  [2]: http://thegenomefactory.blogspot.de/2013/08/paired-end-read-confusion-library.html
  [3]: https://www.biostars.org/p/106291/
  [4]: https://www.biostars.org/p/94246/
  [5]: http://samtools.github.io/hts-specs/SAMv1.pdf
  [6]: https://broadinstitute.github.io/picard/command-line-overview.html
>  So according to my understaning the TLEN number will include the possible introns which means the TLEN would be unsally longer than "actual insert size"? 

Yes, the `TLEN` field won't always be terribly useful in RNAseq. When trying to compute the original fragment sizes it's best to not have spliced fragments. Back when we used tophat2 in our production pipeline, our "insert size estimation" step aligned to the transcriptome to avoid this problem.

> Second, if we are mapping DNA sequences, then the fragment length and "insert size"/"template length" are the same?

This will depend a bit on which fragment you're talking about. See the comment from https://www.biostars.org/u/18713/.

> how Picard tools CollectInsertSizeMetrics actually do to calculate the insert size distribution of a paired-end library, does it only use the TLEN or exclude possible introns?

I've never run that tool on RNAseq data, I'm not sure how useful it would be. I would expect that it's just summarizing the `TLEN` field, so I'd expect some absurdly high mean values.