@article {Scott036061, author = {Erick R. Scott and H. Benjamin Larman and Ali Torkamani and Nicholas J. Schork and Nathan Wineinger and Max Nanis and Ryan Thompson and Reza B. Beheshti Zavareh and Luke L. Lairson and Peter G. Schultz and Andrew I. Su}, title = {RASLseqTools: open-source methods for designing and analyzing RNA-mediated oligonucleotide Annealing, Selection, and, Ligation sequencing (RASL-seq) experiments}, elocation-id = {036061}, year = {2016}, doi = {10.1101/036061}, publisher = {Cold Spring Harbor Laboratory}, abstract = {RNA-mediated oligonucleotide Annealing, Selection, and Ligation (RASL-seq) is a method to measure the expression of hundreds of genes in thousands of samples for a fraction of the cost of competing methods. However, enzymatic inefficiencies of the original protocol and the lack of open source software to design and analyze RASL-seq experiments have limited its widespread adoption. We recently reported an Rnl2-based RASL-seq protocol (RRASL-seq) that offers improved ligation efficiency and a probe decoy strategy to optimize sequencing usage. Here, we describe an open source software package, RASLseqTools, that provides computational methods to design and analyze RASL-seq experiments. Furthermore, using data from a large RRASL-seq experiment, we demonstrate how normalization methods can be used for characterizing and correcting experimental, sequencing, and alignment error. We provide evidence that the three principal predictors of RRASL-seq reproducibility are barcode/probe sequence dissimilarity, sequencing read depth, and normalization strategy. Using dozens of technical and biological replicates across multiple 384-well plates, we find simple normalization strategies yield similar results to more statistically complex methods.}, URL = {https://www.biorxiv.org/content/early/2016/01/07/036061}, eprint = {https://www.biorxiv.org/content/early/2016/01/07/036061.full.pdf}, journal = {bioRxiv} }