Table of Contents
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Many types of small RNA have been characterized, and their biological functions are extremely wide-ranging. The table below describes the different forms and biological functions of small RNAs.
Yao Y, Sun Q. Exploration of small non coding RNAs in wheat (Triticum aestivum L.). Plant Mol Biol. 2012;80(1):67-73.
Clearly, there are many biologically important functions executed by small RNA, and they can be studied by sequencing by simply cutting (for example) the 25-50bp range out of a size selection gel followed by otherwise normal library preparation. Otherwise, all these species share certain qualities that allow sequencing data derived from each to be analyzed in a similar fashion. These qualities can include (but are not limited to):
- Single-end sequencing: if an RNA-species is (for example) 16-25 bp, than paired-end sequencing of any kind provides little (though some) additional data compared to a single end run provided the reads are long enough
- Increased adapter contamination: as implied above, adapter sequence is almost always included in your reads requiring either pre-processing to remove such sequence or alignment adjustment to account for it
- Low-complexity libraries: there are often far fewer members of a category of small RNA in a genome than there are reads in the data, meaning the exact same sequence will occur in multiple reads
- Extensive genomic duplication: there are often many copies of the same sequence of a given small RNA in a genome, meaning most genomic alignments will contain numerous "multi-mappers"
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For "normal" RIP-seq, one usually expects to recover full RNA molecules regardless of where on an RNA molecule the protein was bound, since all of it is 'pulled down' together. However, such protocols generally do not use any chemical or physical means to covalently attach the RNA to the protein, which allows for the possibility that the RNA and protein complexes disassociate and re-associate from each other during sample preparation (there have been published papers that claim this - see here). Moreover, proteins will often bind to specific RNA sequence motifs or positions, and retrieval of the full RNA molecule provides no information about the specific binding site. To accommodate these concerns, methods have been developed to cross-link protein to RNA in a way that leaves a signature of interaction where the protein and RNA actually come into contact. Below is a table of the three methods that modify the RNA in various ways to enable binding site detection by sequencing.
König J, Zarnack K, Luscombe NM, Ule J. Protein-RNA interactions: new genomic technologies and perspectives. Nat Rev Genet. 2011;13(2):77-83.
In our second exercise, we will use a recently developed tool to analyze some sample PARCLIP data to identify specific binding sites of a protein across the entire human transcriptome.
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