MISSSL: Multiple Insert Size Strand-specific Library


Deep transcriptome sequencing is a powerful method for parallel quantitative and qualitative analysis of all mRNAs in a sample. According to a recent comparison of the diverse methods available for transcriptome library preparation [1], the top two approaches are dUTP second-strand marking and the Illumina RNA ligation protocol. A major limitation of the Illumina RNA ligation protocol is the inability to implement paired-end sequencing, although Levin et al. note that modifications to overcome this drawback should be possible. We have adjusted the Illumina RNA ligation protocol to allow for paired-end sequencing and evaluated a C. elegans test library using the methods proposed in Levin et al. Article Supplemental material

Evaluation Code

For the evaluation of our RNA-Seq library, we downloaded the scripts used for the analyses by Levin et al. from the supplementary website and adapted them to the C. elegans genome and our read data. The modified code is available on our FTP server.

Supplementary Data

You can download the data that was used for library evaluation here: Supplemental Table S1: 2011RNABIOL0167R-TS1.xlsx Supplemental Table S2: 2011RNABIOL0167R-TS2.xlsx

You can download the Matlab code used to determine the optimal insert sizes here:

Full alignments and sequenced reads are available on request.


In case of comments, questions etc. feel free to contact Gunnar Rätsch.


[1] Levin JZ, Yassour M, Adiconis X, Nusbaum C, Thompson DA, Friedman N, Gnirke A, Regev A. (2010): Comprehensive comparative analysis of strand-specific RNA sequencing methods.