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- 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"
All of these issues can be taken into account effectively, and in some regards can produce results simpler to understand and evaluate relative to standard gene expression data. Our first exercise will focus on one class of small RNAs, microRNAs, and will use principles that generalize to other interesting small RNAs.
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Similarly, RNA-protein interactions are required for an equally diverse set of biological functions, and hundreds of RNA-binding proteins have been identified. It is frequently interesting to isolate protein-RNA complexes, remove the protein, and sequence the resulting RNA. The methods involve combine components of RNA-seq, because the underlying molecule is RNA, and chromatin immunoprecipitation (ChIP), because the most common mechanism to isolate a protein-RNA complex is with an antibody raised against a fragment of the protein of interest. Below is a sample protocol flow for a RIP-seq experiment.
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 reassociate re-associate from each other during sample preparation (there have been published papers that claim this - see here). Moverover, . 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.
Important Software
For standard RIP-seq, many of the methods already covered in this class are useful since one can expect to recover a full RNA molecule, and the IP and Input samples can be thought of as "conditions" to be compared by differential expression analysis. However, more specific tools do exist, particularly for CLIP-seq and its variants.
Exercise #1: miRNA/small RNA Sequencing and Profiling (miRNA-seq)
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