What are the challenges of RNA-Seq technology?

RNA-seq is a costly and time-intensive procedure. Experiments may take 1.5 to 12 days, depending on the desired goal. Generating libraries for mRNA sequencing is also difficult due to the amount of steps and loss of sample at each step. The RNA must be extracted and reverse transcribed, then processed more in order to generate the sequencing library. The presence of high abundance RNAs requires additional steps to reduce background RNA or enrich mRNAs. RNA-seq technology does not reliably detect allele frequency of mutations. Because coding regions of genes are transcribed into mRNA, RNA-seq is unable to detect any variants that exist in noncoding DNA regions. The complexity of the computational analysis is another limitation because it requires correct mapping of RNA-seq reads to the reference genome. This analysis has to account for mRNA splicing which can be affected by mutations at gene splice sites.