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.