Expression profiling by high throughput sequencing
Summary
RNA-seq is the standard method for profiling gene expression in many biological systems. Due to the wide dynamic range and complex nature of the transcriptome, RNA-seq provides an incomplete characterisation, especially of lowly expressed genes and transcripts. Targeted RNA sequencing (RNA CaptureSeq) focuses sequencing on genes of interest, providing exquisite sensitivity for transcript detection and quantification. However, uses of CaptureSeq have focused on bulk samples and its performance on very small populations of cells is unknown. Here we show CaptureSeq greatly enhances transcriptomic profiling of target genes in ultra-low-input samples and provides equivalent performance to that on bulk samples. We validate the performance of CaptureSeq using multiple probe sets on samples of iPSC-derived cortical neurons. We demonstrate up to 275-fold enrichment for target genes, the detection of 10% additional genes and a greater than 5-fold increase in identified gene isoforms. Analysis of spike-in controls demonstrated CaptureSeq improved both detection sensitivity and expression quantification. Comparison to the CORTECON database of cerebral cortex development revealed CaptureSeq enhanced the identification of sample differentiation stage. CaptureSeq provides sensitive, reliable and quantitative expression measurements on hundreds-to-thousands of target genes from ultra-low-input samples and has the potential to greatly enhance transcriptomic profiling when samples are limiting.
Overall design
Post-capture libraries. Multicentre bulks 58 replicate each capture panel (TF capture, NF capture). Two sets of oligonucleotide capture probes were designed for synthesis as Roche NimbleGen SeqCap Libraries. The first targeted human neurological disease risk loci and candidate genes (NeuroGWAS) ,while the second targeted human transcription factor gene (hereafter referred to as the NF and TF captures respectively). 24 mini-bulk samples: 8 replicates each for each capture condition (NF panel 850ng, 850ng TF panel, 150ng TF panel). PreCapture libraries: Multicentre bulks: 58 replicates. Minibulks: 8 replicates for each capture panel (TF capture, NF capture).
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