The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis.
Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom.
Betty Y
BY
Chung
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom.
Thomas J
TJ
Hardcastle
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.
Joshua D
JD
Jones
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.
Nerea
N
Irigoyen
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.
Andrew E
AE
Firth
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom.
David C
DC
Baulcombe
Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.
Ian
I
Brierley
D016825:Chlamydomonas reinhardtii / Q000235:genetics; D019295:Computational Biology; D004720:Endonucleases / Q000378:metabolism*; D016366:Open Reading Frames; D012270:Ribosomes / Q000378:metabolism*; D012984:Software
D004720:Endonucleases
Chlamydomonas; duplex-specific nuclease; mouse; ribosome profiling; translation