I want to know how a skipped region in the reference, or N in the CIGAR string, looks in the alignment. To try and explain what I mean I use the example provided from the SAM format specification (http://genome.sph.umich.edu/wiki/SAM), which does not include an N example:

```
Ref + read
RefPos:     1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17 18 19
Reference:  C  C  A  T  A  C  T  G  A  A  C  T  G  A  C  T  A  A  C
Read: ACTAGAATGGCT

Alignment
RefPos:     1  2  3  4  5  6  7     8  9 10 11 12 13 14 15 16 17 18 19
Reference:  C  C  A  T  A  C  T     G  A  A  C  T  G  A  C  T  A  A  C
Read:                   A  C  T  A  G  A  A     T  G  G  C  T

Cigar:
POS: 5
CIGAR: 3M1I3M1D5M
```

---

Now, in position 11 there is an insertion in the reference sequence. However, I would think that you can't distinguish between a skipped region or an insertion in the reference. Therefore the CIGAR string could also have been `3M1I3M1N5M`

So how is it the alignment of a skipped region or an insertion in the reference sequence different? Is it only a skipped region if the C in position 11 is an N?
There's no *a priori* way to always distinguish between deletions (D CIGAR operations) and splicing (N CIGAR operations). In practice, most RNAseq aligners (e.g., tophat2 and STAR) have parameters with semi-arbitrary thresholds for the minimum intron size or maximum deletion size. In the case of STAR, any gap less than `alignIntronMin` (21 bases last I looked) is considered a deletion. I can't recall exactly what the tophat2 option for this is off-hand, but it's in there somewhere.

It's probably worth pointing out that the default values for these might be worth changing in some cases. I suspect that if someone were interested in splicing changes in cancer cells where there are a bunch of deletions that these parameters might need some tweaking (though presumably one would do WGS or WES alongside).