Expression Profiling with 3‘-Libraries
My last week’s blog article was about expression profiling with mRNA-Seq libraries and about the required sequencing depth of this protocol. But there are other possibilities for expression profiling, and today I especially want to highlight the 3’-fragment library protocol.
The big advantage of this protocol is that it provides a much higher resolution than mRNA-Seq does. The reason is that within mRNA-Seq the average transcript is represented by approx. 10-25 reads that cover the whole transcript, while with the 3’-fragment protocol only one read is generated per transcript. The derived reads from a 3’-end library map to the 3’-end of the transcripts and expression differences are easily collected by just counting the reads that map to a specific reference transcript.
The 10-25-fold higher resolution comes along with considerably reduced projects costs as 10-25-fold less sequencing is required to obtain a similar depth of the analysis. Or in other words: When analyzing the same number of samples per channel the 10-25 fold higher resolution allows the scientist to even look at very low expressed genes with reliable statistical evidence.
Of course the mRNA-Seq protocol is needed in case other analysis shall follow, like the study of alternative transcripts, or fusion genes. But this is anyway a completely different story as these applications need an even higher sequencing depth than expression profiling with mRNA-Seq does require.
As a conclusion I think it is definitely worth to evaluate this protocol when having in mind an expression profiling experiment. And we would be delighted if you share your thoughts on this with us and the other blog readers.