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 1000’s of samples and amplicon analysis is making it possible to run 10,000’s of samples in a single experiment (we’ve run a HiSeq flowcell with 12288 samples on it, 1536 per lane using Fluidigm – currently unpublished).
The next frontier looks like it could be using the same techniques to target a portion of the transcriptome, again allowing many 1000’s or 10,000’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 Illumina’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.
Hi James,
ReplyDeleteExcellent Blog Post! I too have been interested in the coming of age of Targeted RNA-sequencing, and recently we have been focusing our efforts toward the need of targeted RNA-sequencing assays to be1) quantitative, 2) reproducible in quantitation, and 3) at a drastically reduced need for sequencing depth for large dynamic ranges of templates. All of these items or critical for adoption of routine use of targeted RNA-sequencing based diagnostics in the clinical setting. In particular, point (3) above is extremely important and limits the sample multiplexing level of targeted RNA-sequencing assays, which drastically increases the cost on a per sample basis in return.
The work in the study in the URL attached with this comment post represents our recently published approach to make targeted RNA-sequencing highly quantitative yet removes the need to over-sequence abundant targets; all with NO signal compression in the measured dynamic range of templates! The publication presents Ion Torrent data, but since then we have shown this approach works well on Illumina hiseq and miseq. Our unpublished data shows our results are concordant between the two platforms as well. These latter concordance studies will be out in 2014. All of these studies are part of a larger FDA initiative for concordance between RNA sequencing studies.
I realize this may be a bit of bombing your post but linking to the article, but at the same time, it is directly relevant to the cost-effectiveness question you raise at the end.
Also, I too think genomics may just be the next best thing since sliced bread. :-)
Regards, -Tom Blomquist
That was great to know!.. RNA-Seq is a recently developed approach to transcriptome profiling that uses deep-sequencing technologies. Studies using this method have already altered our view of the extent and complexity of eukaryotic transcriptomes. RNA-Seq and Small RNA Sequencing provides a far more precise measurement of levels of transcripts and their isoforms than other methods. This article describes the RNA-Seq approach, the challenges associated with its application, and the advances made so far in characterizing several eukaryote transcriptomes. Learn more.. Thanks
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