However, most NGS library prep kits fail to adequately capture RNAs of a diverse range of sizes from limited and low-quality samples, such as formalin-fixed, paraffin-embedded (FFPE) samples. This is due to the efficiency of the enzyme that is responsible for adding adapters required for library amplification. Specifically, reactions are prone to nucleotide bias – some fragments with certain nucleotides at the 3’ end will be ligated with better efficiency than other fragments, resulting in a biased library.
Consequently, researchers who want to profile both small and large RNAs need to create two separate libraries. One library would contain only long RNAs, and the other would contain only short ones, while RNAs of intermediate size would get lost altogether. Each library would need to be sequenced separately and then researchers would need to use bioinformatics to normalize gene expression between the two datasets. This process is not only time consuming, ...
