The Captureseq Technique Is More Accurate for Low Expressing Genes and Long Non-Coding RNAs

This somewhat arbitrary limit distinguishes lncRNAs from small regulatory RNAs such as microRNAs (miRNAs), short interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs. LncRNAs have been found to be involved in numerous biological roles including imprinting, epigenetic gene regulation, cell cycle and apoptosis, and metastasis and prognosis in solid tumors. Most lncRNAs are expressed only in a few cells rather than whole tissues, or they are expressed at very low levels, making them difficult to study.

RNA sequencing (RNAseq) samples the majority of expressed genes infrequently, owing to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes. This results in sparse sequencing coverage that can hinder robust isoform assembly and quantification. RNA capture sequencing (CaptureSeq) addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for targeted sequencing. The method involves enriching transcripts of interest by hybridizing them to magnetic bead-linked oligonucleotides that are tiled across the region of interest, allowing for targeted purification, multiplexed library preparation, and RNA sequencing at a high depth.

Investigators at the Garvan Institute of Medical Research (Sydney, Australia) recently compared quantitative real time-PCR (qRT-PCR), RNA-sequencing (RNAseq), and capture sequencing (CaptureSeq) in terms of their ability to assemble and quantify lncRNAs and novel coding exons across 20 human tissues.

They reported in the March 9, 2015, online edition of the journal Nature Methods that CaptureSeq achieved eightfold better sequence coverage for all standard concentrations tested, corresponding to the assembly of as few as 1,550 transcripts in the input RNA. In contrast, RNAseq could not reliably detect low standard concentrations, precluding the measurement of low-abundance standards. In the human leukemia cell line K562, an estimated 42.1% of RNA transcripts were better quantified using CaptureSeq. RNAseq and CaptureSeq performed similarly for 53.2% of transcripts.

While RNAseq performed better than CaptureSeq for the most highly expressed 4.6% of transcripts enriched for housekeeping, structural, and metabolic genes, genes with low expression in K562 cells for which CaptureSeq provided superior quantitative accuracy were enriched for transcription factors and genes associated with cancer or other human diseases.
Finally, the investigators identified 13,796 loci that generated 45,399 lncRNA isoforms, of which 27,596 were previously unknown, with 20.6% more exons and 13.5% more introns compared with previous annotations.

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Garvan Institute of Medical Research