Following up on my [previous question](https://www.biostars.org/p/209854/), I have inspected the length distribution of the first exons of spliced transcripts in GENCODE26.

I still find it surprising that some are tiny, often starting at the translation start site, while the transcript is not annotated as "processed".  A list of all first spliced exons shorter than 4 nt can be found at the bottom of the following page <https://cdn.rawgit.com/charles-plessy/gencode-feature-lengths/d55347a9/gencode-feature-lengths.html> and the source code generating it is also available [on GitHub](https://github.com/charles-plessy/gencode-feature-lengths).

Is there a better explanation than just imperfections in processing pipelines or annotations ?

The reason I ask is that I would like to know if there is a lower limit of first exons at the time of RNA splicing.  (And the reason behind the reason is that I have a sequence library from which I can infer the length of first exons, and I would like to test if the results are reliable).

![Length of first spliced exons](https://raw.githubusercontent.com/charles-plessy/gencode-feature-lengths/960c0b25eae5c52bf07dc8c6c3ea3862174b0aa1/gencode-feature-lengths_files/figure-html/first-microexons-1.png)
This is an interesting analysis of the GENCODE v26 geneset, that highlights potential idiosyncrasies in the annotation process.  There are however some important points to consider when interpreting these results.

While the current geneset contains first 'exons' with less than 20nt, these do not necessarily represent the full-length of the exon. Indeed, these first exons may not actually represent the transcriptional start of the associated transcript. This is because manually annotated GENCODE transcripts are extended to the limit of the supporting evidence, which typically consists of ESTs and cDNAs. If this evidence doesn't extend to the actual TSS, then the annotated transcript will essentially be truncated at the 5' end.  This appears to be the case for most of the 'micro first exons' identified here, as they coincide with longer exons in other transcript isoforms for the same gene.

Although the annotation of such short exons may be accurate, their actual value within the geneset may be limited. It may be best therefore to exclude such transcripts from any analysis of RNA splicing.  On behalf of GENCODE, we welcome any comments or questions, which can be sent to either [HAVANA][1] or [GENCODE][2].


  [1]: mailto:havana-help@sanger.ac.uk
  [2]: mailto:gencode-help@sanger.ac.uk