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Nat Protoc. 2016 Sep;11(9):1650-67. doi: 10.1038/nprot.2016.095. Epub 2016 Aug 11.

Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown.

Author information

1
Center for Computational Biology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.
2
Department of Computer Science, Whiting School of Engineering, Johns Hopkins University, Baltimore, Maryland, USA.
3
Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
4
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA.

Abstract

High-throughput sequencing of mRNA (RNA-seq) has become the standard method for measuring and comparing the levels of gene expression in a wide variety of species and conditions. RNA-seq experiments generate very large, complex data sets that demand fast, accurate and flexible software to reduce the raw read data to comprehensible results. HISAT (hierarchical indexing for spliced alignment of transcripts), StringTie and Ballgown are free, open-source software tools for comprehensive analysis of RNA-seq experiments. Together, they allow scientists to align reads to a genome, assemble transcripts including novel splice variants, compute the abundance of these transcripts in each sample and compare experiments to identify differentially expressed genes and transcripts. This protocol describes all the steps necessary to process a large set of raw sequencing reads and create lists of gene transcripts, expression levels, and differentially expressed genes and transcripts. The protocol's execution time depends on the computing resources, but it typically takes under 45 min of computer time. HISAT, StringTie and Ballgown are available from http://ccb.jhu.edu/software.shtml.

PMID:
27560171
PMCID:
PMC5032908
[Available on 2017-03-01]
DOI:
10.1038/nprot.2016.095
[Indexed for MEDLINE]
Free PMC Article

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