I have a question regarding stranded RNA-Seq from Illumina TruSeq. I'm comparing some stranded RNA-Seq runs from SOLID and Illumina. I have found that while SOLID stranded protocol is perfect (literally no reads aligning to antisense strand), I found quite a lot reads in antisense strand in Illumina sample. Still sense strand reads are enriched (~20 times more reads in sense strand).

The reads from antisense strand are aligned uniquely (MAPQ>100, mapped with STAR), but I don't think it's real expression from antisense strand, as the exon-intron structure of antisense reads is similar to exon-intro structure from sense. But I see huge enrichment 10-20x) in Illumina

Is TruSeq designed to enrich for sense strand or is it suppose to be perfectly stranded protocol as SOLID was?

Is it possible that something went wrong during library prep?

<img alt="< image not found >" src="https://imagizer.imageshack.us/v2/692x523q90/661/3gigpd.png" style="height:523px; width:692px" />
I have found highly variable sense-strand read enrichment (6.39-27.05; median: 9.85) in our RNA-Seq stranded data (6 time points with 3 RNA fractions in colours). I have ignored exons having any overlap with known antisense exons.

Overall, red RNA fraction tends to have higher sense-strand read enrichment that blue/orange. **Any clue why there is such high variability in enrichment between time points / RNA fractions?**

I have obtained slightly lower enrichment for genes (5.98-24.07; median: 8.74). As these data is from zebrafish, for which the annotation is far from being perfect, I suspect many antisense genes / exons are simply not annotated.

More about methodology [here][1].

<img alt="< image not found >" src="http://i.imgur.com/PQza8Wv.png" style="height:371px; width:600px" />

 [1]: http://bioinfoexpert.com/?p=447