Week 3 Flashcards
Sequencing genomes
resulted in a shift from studying single or a few genes to studying all genes simultaneously
proteome and transcriptome
Genome
all DNA and identification of all DNA elements (transcriprion units)
Transcriptome
all transcripts expressed (list plus analysis of expression)
Proteome
all proteins expressed (list plus analysis and modification)
Large scale ORF finder
Looking for open reading frames in the bacteria
Simple for bacteria because of the fact that DNA contains the coding region that is not interrupted.
So you can go from DNA to the protein coding capacity of that DNA very simply.
We can’t do the same for the eukaryotic DNA.
Eukaryotes and ORF finders
not for most eukaryotes, we can’t go from the eukaryotic genome to the eukaryotic proteome that simply
Splicing
We need the transciptome to get the proteome of the genome.
transcriptome is
all expressed RNA: mRNA rRNA tRNA siRNA miRNA non coding RNA snRNA crRNA snoRNA
eukaryotic mRNA
exstensively processed
5’ prima cap
AUG first codon of ORF
Messenger RNAs are processed with the additon of a poly-A-tail that helps us annotate the proteosome
reverse transcriptase
the DNA copy is made with reverse trancriptase which requires a DNA primer. A common approach is to use an oligo dT primer that hybridizes with the poly A tail. therefore the total transcriptome is not represented
Before nanopore only DNA could be sequenced so RNA always had to be turned into a complementary DNA copy.
post translational processing
a barrier to annotating the genome
a primary transcript is processed, splicing, poly-a-tail and cap
Therefore anytime we make a complementary DNA copy we’re making a complementary copy of the mature mRNA after the intronic sequences are removed.
A large amount of the genome is not expressed: intragenic regions which are not trasncirbed, intronic regions that are transcribed but spliced out.
post translational processing
a barrier to annotating the genome
a primary transcript is processed, splicing, poly-a-tail and cap
Alternative Splicing
Genes undergo alternative splicing, when you align different cDNA sequences to the genome you find that some genes that these aligments are quite different from one cDNA to another
indicating that they came from transcripts that have undergone alternative splicing
This gene produces six distinct messenger rna transcripts.
That encode three distinct polypeptides.
When you align this sequence to drosophila DNA you ifnd six different patterns of alignments due to six different splicing patterns of the mRNA transcripts.
Alternaitve splicing increases the number of proteins that can be encoded by a single gene.
Types of splicing
alternative poly-a-tail sites alternative promoters Exon included or excluded Mutually exclusive inclusion. Alternative 5’ splice sites. Alternative 3’ splice sites. Retained intron
In some messages splicing occurs such that the intron remains in the mature mRNA, in other the mature mRNA the intron is removed.
RNA seq Two major goals
Count the relative number of transcripts in the sample.
Determine the structure of the transcripts in the sample.
Often done after they’ve converted the RNA to complementary DNA and sequenced the complementary DNA.
How do we get distinct cell types
differential gene expression
sc RNA seq goals
To determine the poly A+ transcriptome of individual cells
Useful in the study of development and human disease