Illumina and Life Technologies are both launching targeted RNA-seq applications which are likely to become standard tools for many labs; if the price is right.
The ability to target a portion of the genome has revolutionised next-generation sequencing experiments. The analysis of exomes has exploded, custom panels for exome-style pull-down are being used to great effect in 1000â€™s of samples and amplicon analysis is making it possible to run 10,000â€™s of samples in a single experiment (weâ€™ve run a HiSeq flowcell with 12288 samples on it, 1536 per lane using Fluidigm â€“ currently unpublished).
The next frontier looks like it could be using the same techniques to target a portion of the transcriptome, again allowing many 1000â€™s or 10,000â€™s of samples to be analysed in a single experiment. These technologies are likely to replace real-time PCR for mid- to high-plex studies. Anyone that has tried to run a few 100 TaqMan or SYBR assays on their 96-well qPCR machine will see the potential. And users with BioMark, TLDA, Wafergen, and other high-throughput qPCR systems will see the potential of using just one analysis method (NGS) as their primary data collection tool.
Illumina: The new TruSeq Targeted RNA Kits use just 50ng of total RNA users synthesise cDNA, which is the input for a golden-gate extension-ligation assay. Pairs of oligos are designed to specific transcripts or splice-junctions, thee are hybridised to cDNA, extended with polymerase and ligated to form a molecule that can be amplified in a universal barcoded PCR.
Users can design panels of up to 1000 assays to genes, isoforms splice junctions and coding SNPs. The assay can be completed in one day with a very quick MiSeq run allowing results to be ready in 24 hours.
Up to 2000 assays can be run in a single experiment, and Illuminaâ€™s DesignStudio allows you to determine the number of MiSeq runs required to achieve around 1000 fold coverage of each RNA. Sequencing is performed using a very quick single-end 50bp run, allowing the complete workflow, RNA to data, to be finished in under 24 hours.
Life Technologies: The Ion AmpliSeq RNA technology creates a library of gene specific assays similar to TaqMan assays but without the probes. PCR amplification from cDNA allows counting of the transcripts. At the moment 12-300 genes can be assayed at once in up to 96 samples. They have a handy flyer here.
There is a Cancer panel targeting 50 genes, an apoptosis panel with 267 genes and you can create custom panels from 21,000 assays.
The method uses 500pg of unfixed RNA or 5ng of FFPE RNA as input and Life suggest that the whole process can be carried out in about 15 hours, spread over 1.5 days
Who will use them: Both systems will allow users to run potentially 10,000’s of qPCR assays in a single sequencing experiment. The cost savings in TaqMan assays or primer sets for SYBR-green qPCR are likely to be high, but the cost per sample may keep the technology out of reach for many labs. Amplicons from DNA are not cheap using TSCA or AmpliSeq and many labs are experimenting with other ways to get these analysed on MiSeq etc more cost-effectively.