Cuffdiff update for differential expression analysis of RNA-Seq data

Differential analysis of gene and transcript expression using high-throughput RNA sequencing (RNA-seq) is complicated by several sources of measurement variability and poses numerous statistical challenges.

A team led by researchers at the University of California Berkeley has released and update to Cuffdiff which is their approach to differential expression analysis in Cufflinks. The update, Cuffdiff 2, is an algorithm that estimates expression at transcript-level resolution and controls for variability evident across replicate libraries. Cuffdiff 2 robustly identifies differentially expressed transcripts and genes and reveals differential splicing and promoter-preference changes.

In this article, they demonstrate the accuracy of their approach through differential analysis of lung fibroblasts in response to loss of the developmental transcription factor HOXA1, which they show is required for lung fibroblast and HeLa cell cycle progression. Loss of HOXA1 results in significant expression level changes in thousands of individual transcripts, along with isoform switching events in key regulators of the cell cycle. Cuffdiff 2 performs robust differential analysis in RNA-seq experiments at transcript resolution, revealing a layer of regulation not readily observable with other high-throughput technologies.

Availability – Cuffdiff 2 is available at:

Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L.(2012) Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol [Epub ahead of print]. [abstract]

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