Editing BioMicroCenter:Research (section)

=== Testing of NuGen’s  SPIA Technology for Illumina Sequencing - [http://web.mit.edu/biology/www/facultyareas/facresearch/burge.html BURGE LAB] - [http://web.mit.edu/be BE] - [http://web.mit.edu/biology Biology] - [http://web.mit.edu/ki KI] === 
One of the key limitations of RNA-sequencing is the relatively large amounts of RNA required for each sample. While recent protocols have reduced the amount of total RNA input down below 1g, the initial protocols required 5-10g of material. This is several orders of magnitude higher then we routinely use for microarray analysis. 
While most microarray labeling protocols are inappropriate for Illumina sequencing in that they use cRNA as their labeled materials, the NuGen kits used by the BioMicro Center since 2009 are unique in that they use amplified cDNA which has several benefits to microarray analysis. We were particularly interested in the ability of NuGen to handle amounts of RNA in the sub-nanomolar range which could allow next-generation sequencing of RNA from single cells. In order to test the viability of this approach, we established a collaboration with NuGen and with Dr. Chris Burge of the Biology and Biological Engineering departments. <BR><BR>
To establish the robustness of the NuGen system for RNA-seq, two mRNA samples were isolated from control and UPF-1 knockdown cells and were prepared either with the NuGen kit (by NuGen technicians) or with standard RNA-seq methods from Illumina (prepared in the Burge lab). The NuGen samples were prepared across a variety of concentrations, and seven paired-end libraries were run.  Differential error rates, coverage, sensitivity and differential expression were all calculated by the Burge Lab. <BR><BR> 
Our results demonstrated that the NuGen kit, unfortunately, has a number of issues that are concerning in the RNA-seq environment. Analysis of coverage showed very uneven coverage of exons, likely due to the semi-random-nonamers that NuGen uses in preparing the cDNA. In addition, the level of noise introduced in differential expression was quite large, at least for an experiment with subtle changes in expression.  Our results have discouraged us from focusing on NuGen protocols for looking at RNA-seq data. It has even raised questions to us about the quality of the NuGen kit for exon array analysis, though other whole transcriptome amplifications are probably no better.