ncRNAseq: simple modifications to RNA-seq library preparation allow recovery and analysis of mid-sized non-coding RNAs

Despite their abundance, mid-sized RNAs (30-300 nt) have not been extensively studied by high-throughput sequencing, mostly due to selective loss in library preparation. The authors propose simple and inexpensive modifications to the Illumina TruSeq protocol (ncRNAseq), allowing the capture and sequencing of mid-sized non-coding RNAs without detriment to the coverage of coding mRNAs. This protocol is coupled with a two-step alignment: a pre-alignment to a curated non-coding genome, passing only the non-mapping reads to a standard genomic alignment. ncRNAseq correctly assigns the highest read-numbers to established abundant non-coding RNAs and correctly identifies cytosolic and nuclear enrichment of known non-coding RNAs in two cell lines. METHOD SUMMARY In order to retain RNAs shorter than 300 nt and the cDNAs they give rise to, the authors reduce the fractionation time of the RNA and increase the efficiency of four precipitation steps in the Illumina TruSeq protocol (ncRNAseq): twice pre-ligation by adding isopropanol to AMPure beads and twice post-ligation by using a higher AMPure-to-sample ratio. The resulting sequencing reads are aligned first to a custom non-redundant curated 'non-coding genome', which allows the user to exert control, for example, on amalgamation of multi-copy genes or separation of non-coding intronic gene counts from coding host gene counts. The non-mapping reads are then passed to a standard genomic alignment in a lossless manner.