Small RNA sequencing

Small RNA sequencing (Small RNA-Seq) is a type of RNA sequencing based on the use of NGS technologies that allows to isolate and get information about noncoding RNA molecules in order to evaluate and discover new forms of small RNA and to predict their possible functions. By using this technique, it is possible to discriminate small RNAs from the larger RNA family to better understand their functions in the cell and in gene expression. Small RNA-Seq can analyze thousands of small RNA molecules with a high throughput and specificity. The greatest advantage of using RNA-seq is represented by the possibility of generating libraries of RNA fragments starting from the whole RNA content of a cell.
Introduction.
Small RNAs are noncoding RNA molecules between 20 and 200 nucleotide in length. The item "small RNA" is a rather arbitrary term, which is vaguely defined based on its length comparing with regular RNA such as messenger RNA (mRNA). Previously bacterial short regulatory RNAs have been referred to as small RNAs, but they are not related to eukaryotic small RNAs. 
Small RNAs include several different classes of noncoding RNAs, depending on their sizes and functions: snRNA, snoRNA, scRNA, piRNA, miRNA,YRNA, tsRNA, rsRNA, and siRNA. Their functions go from RNAi (specific for endogenously expressed miRNA and exogenously derived siRNA), RNA processing and modification, gene silencing (i.g. X chromosome inactivation by Xist RNA), epigenetics modifications, protein stability and transport.
Small RNA sequencing.
Purification.
This step is very critical and important for any molecular-based technique since it ensures that the small RNA fragments found in the samples to be analyzed are characterized by a good level of purity and quality. There are different purification methods that can be used, based on the purposes of the experiment: 
Once small RNAs have been isolated, it is important to quantify them and to evaluate the quality of the purification. There are two different methods to do this: 
Library preparation and amplification.
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. 
Sequencing.
Depending on the purpose of the analysis, RNA-seq can be performed using different approaches:
Data analysis and storage.
The final step regards analysis of data and storage: after obtaining the sequencing reads, UMI and index sequences are automatically removed from the reads and their quality is analyzed by PHRED (software able to evaluate the quality of the sequencing process); reads can then be mapped or aligned to a reference genome in order to extract information about their similarity: reads having the same length, sequence and UMI are considered as equal and are removed from the hit list. Indeed, the number of different UMIs for a given small RNA sequence reflects its copy number. 
The small RNAs are finally quantified by assigning molecules to transcript annotations from different databases (Mirbase, GtRNAdb and Gencode).
Applications.
Small RNA sequencing can be useful for: