RT Journal Article SR Electronic T1 RASLseqTools: open-source methods for designing and analyzing RNA-mediated oligonucleotide Annealing, Selection, and, Ligation sequencing (RASL-seq) experiments JF bioRxiv FD Cold Spring Harbor Laboratory SP 036061 DO 10.1101/036061 A1 Erick R. Scott A1 H. Benjamin Larman A1 Ali Torkamani A1 Nicholas J. Schork A1 Nathan Wineinger A1 Max Nanis A1 Ryan Thompson A1 Reza B. Beheshti Zavareh A1 Luke L. Lairson A1 Peter G. Schultz A1 Andrew I. Su YR 2016 UL http://biorxiv.org/content/early/2016/01/07/036061.abstract AB 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.