Bimodal insert size distribution

[Pepe]

Hi,

we have used the RNAseq Illumina protocol to make about a dozen paired-end libraries.

Once sequenced and aligned to the reference genome I plot the distribution of insert sizes and I see a bimodal distribution. One peak corresponding to the insert size I'm expecting from the band size we cut (around 220 bp) and the other peak at a smaller size (around 100bp). See attached.

I see different sizes of that peak at 100bp in some of the libraries and not at all in others, which makes me think it happened during the library preparation.

I look at the reads under both peaks and they look very similar to me (many perfect matches, evenly distributed across the genome...)

I was expecting bigger insert sized due to the alignment of the (RNA) reads to the genomic sequence (no introns), but not smaller.

Any ideas on what's going on?

Thanks in advance

[natstreet]

I realise this is a long time past the original post - but could you tell me how you produced the graph you attached?