RNA Sequencing Flashcards
What are the important elements involved in splicing?
snRNPs come together to make the spliceosome
Donor and receptor sites on DNA have specific residues (donor pretty much always has GT and acceptor has AG)
Branchpoint is also an important site the spliceosome needs to recognize
How can RNA sequencing increase diagnostic yield?
Pairing whole genome sequencing with whole transcriptome sequencing
Identifying expression changes associated with non-coding variants (promoters and UTRs)
Identifying loss of mRNA expression in candidate disease genes
How can RNA sequencing help with variant interpretation?
Seeing what transcripts are produced by various tissues
Analyzing tissue specificity of normal and abnormal splicing changes
Clarifying if Copy Number Variants (CNVs) are in tandem
Allele specific expression - is an abnormal RNA transcript exclusively produced from the variant allele?
What are different names given to important splice sites?
Essential/canonical splice sites at +/-1, 2 positions
Conserved splice sites at +/- 3, 4, 5, 6 positions and first/last nucleotide of exon
What are some of the effects on RNA with altered splicing?
Exon skipping, partial intron inclusion and exon skipping, cryptic exon inclusion, partial exon deletion
What are some possible effects on protein caused by altered splicing?
truncated protein, in-frame deletion/insertion of AA, RNA degradation, loss of translation
How are splicing changes measured?
With a metric called percent-spliced in (PSI)
What are some challenges with interpreting RNA splicing data?
One DNA variant can cause one or more RNA effects
Each RNA effect can have a different impact on the protein
DNA variants can altar splicing to different degrees from complete to not at all
Presence of normal, alternative splicing
Differences in expression and transcripts between blood and other tissues
