Many of the NGS sequencing protocols rely on the production of a genomic library that contains thousands of fragments of the target nucleic acids that will then be sequenced by proper technologies. According to the sequencing methods to be used, libraries can be created differently (in the case of the Ion Torrent technology RNA fragments are directly attached to a magnetic bead through an adapter, while for Illumina sequencing, the RNA fragments are firstly ligated to the adapters and then attached to the surface of a plate): generally, universal adapters A and B (containing well known sequences comprehensive of Unique Molecular Identifiers that are used to quantify small RNAs in a sample and sample indexing that allows to discriminate between different RNA molecules deriving from different samples) are ligated to the 5 and 3 ends of the RNA fragments thanks to the activity of the T4 RNA ligase 2 truncated. After the adapters are ligated to both ends of the small RNAs, retrotranscription occurs producing complementary DNA molecules (cDNAs) which will be, eventually, amplified by different amplification techniques depending on the sequencing protocol that is being followed (Ion Torrent exploits the emulsion PCR, while Illumina requires a bridge PCR) in order to obtain up to billions of amplicons to be sequenced. Besides the regular PCR mix, masking oligonucleotides targeting 5.8s rRNA are added to increase sensitivity to small RNA targets and to improve the amplification results. Caution has to be used, as RNA samples are prone to degradation, and further improvement of this technique should be oriented towards the elimination of adapter dimers. Some specific RNA modifications (such as 5′ hydroxyl (5′-OH), 3′-phosphate (3′-P) and 2′,3′-cyclic phosphate (2′3′-cP)) can block the adapter ligation process, while some other RNA modifications ( such as m1A, m3C, m1G and m22G) can interfere with reverse transcription process. Small RNA bearing one or more of these modifications are often inefficiently and incompletely converted into cDNAs, leading to challenges with their detection and quantitation by deep sequencing, which can be overcome by enzyme (such as PNK and AlkB) pre-treatment.