Abstract
Different
technologies support researchers in probing the transcriptome. The choice among
these technologies is guided in part by the balance between the amount of data one
wishes to obtain for a given sample and the number of samples being tested. Typically,
these parameters are inversely correlated. At the opposite ends of this
spectrum, deep RNA sequencing and qPCR yield in depth data for tens of samples starting
at a total cost of at least 300€/sample or very directed information for
thousands of samples at a cost below 10€ per sample, respectively. We here
present HTPathwaySeq, a technology situated in the middle of this spectrum,
tailored towards researchers looking for maximal molecular insights for their
in vitro studies.
At a cost
below 100 EUR/sample, HTPathwaySeq processes 384 cell lysates with RNA seq to
generate expression data analyzed at pathway level. Our data shows that shallow
sequencing of crude cell lysates reproducibly detects over 5000 genes with at
least 10 reads. Subsampling of deep sequencing datasets demonstrated that differential pathway
analysis is largely unaffected when reducing the number of genes to this level.
Consequently, reliable pathway insights can be obtained at high throughput and
relatively low cost while not being limited to a predefined set of genes or
pathways. In cell perturbation screenings (small molecules, RNAi, antisense or
CRISPR), HTPathwaySeq can provide in depth information on the mode of action
underlying the induced cellular phenotypes as well as molecular similarity
scores to identify those perturbations acting similar to a reference condition
or via shared molecular mechanisms. We will show results from a lead compound
dose response study, illustrating the potential of HTPathwaySeq.
