RNA sequencing (RNA-seq) experiments now span hundreds to thousands of samples. A source of frustration for investigators analyzing a given dataset is the inability to rapidly and reproducibly align its samples jointly. Current spliced alignment software is designed to analyze each sample separately. Consequently, no information is gained from analyzing multiple samples together, and it is difficult to reproduce the exact analysis without access to original computing resources. Researchers at Johns Hopkins University have devloped Rail-RNA, a cloud-enabled spliced aligner that analyzes many samples at once. Rail-RNA eliminates redundant work across samples, making it more efficient as samples are added. For many samples, Rail-RNA is more accurate than annotation-assisted aligners. We use Rail-RNA to align 666 RNA-seq samples from the GEUVADIS project on Amazon Web Services in 12 hours for US$0.69 per sample. Rail-RNA produces alignments and base-resolution bigWig coverage files, ready for use with downstream packages for reproducible statistical analysis. We identify 290,416 expressed regions in the GEUVADIS samples, including 21,224 that map to intergenic sequence. We show that these regions show consistent patterns of variation across populations and with respect to known technological confounders. We identify expressed regions in the GEUVADIS samples and show that both annotated and unannotated (novel) expressed regions exhibit consistent patterns of variation across populations and with respect to known confounders.
Rail-RNA eliminates redundant alignment work by first aggregating reads by nucleotide sequence and, where appropriate, aligning only unique read sequences to the genome. Read sequences that do not align perfectly to the genome are divided into overlapping segments, and after these segments are aggregated, each unique segment is aligned to the genome exactly once. Rail-RNA then gathers all segment alignments belonging to a read sequence and searches for introns within each unique read sequence.
Availability – Rail-RNA is open-source software available at http://rail.bio