2010: luminometers OR sequencers?

ResearchBlogging.orgNext generation sequencing (solexa, illumina, 454) is offering a new opportunity for the design of multiplexed reporter assays. With the notable 2007 exception of Brainbow (in which however, it was not possible to discriminate the origin of the 90 or more observed fluorescent colors because they come from random recombination), simply the co-detection of more than three fluorescent proteins is very challenging in real life because of spectral overlapping and other shortcomings. Early in 2008, I blogged about a new method to detect more than 30 reporters simultaneously: instead of coding for a protein, each reporter was coding for a very similar mRNA. The difference between each mRNA was just a 5-nt lenght: i.e. reporter mRNA #1 was 100 nt long, mRNA #2 was 105 nt, mRNA #3 was 110 nt and so on. Once transfected, the library of reporters was reverse-transcribed with the same primer pairs and then analyzed and semi-quantitated by capillary electrophoresis. Today, forget electrophoresis, and think parallel sequencing. The same plasmid backbone can be coupled to an oligo barcode!

The work of Patwardhan et al. open this way: at Washington, they were bothering about the effect of each possible mutation in a promoter, one base at a time. By means of synthetic saturation mutagenesis, mutant promoters were obtained by parallel solid synthesis and then released into solution, resulting in a complex library. The trick was that each oligo in the library was designed to include a unique barcode sequence downstream of the promoter's transcription start site. One library aliquot was used in an in vitro transcription reaction to make RNA from the promoter (subsequently reversely transcribed). The second aliquot was amplified using PCR only. High-throughput illumina sequencing has been carried out and quantitative comparison between the RNA barcode and the DNA barcode was used to give a measure of promoter effectiveness in a way that seems to me more clean than our classical beta-gal normalization. Ok, at the moment the technology is applied only in vitro, but the concept is clear: next generation sequencing can help the reportergenomic community. Give me a barcode plasmid and I'm going to make the reporter mouse.

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Patwardhan, R., Lee, C., Litvin, O., Young, D., Pe'er, D., & Shendure, J. (2009). High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis Nature Biotechnology, 27 (12), 1173-1175 DOI: 10.1038/nbt.1589

another barcoding strategy was blogged on March 2008.