What is a reference genome
Based on 10 individuals
Pangenone
Assembled from lots of samples
De novo assembly
Assembles overlapping reads into larger contings
Clever PCR to confirm deletions
Two PCRs are designed: WT‑specific PCR uses a primer inside the deleted region, so it only amplifies wild‑type DNA. Deletion‑specific PCR uses primers flanking the breakpoints, which only amplify when the large region is deleted (short distance). Presence of both bands indicates a heterozygous deletion.
How do we know which variants are disease causing
Where in the gene: Exon/Intron/Intergenic
• What could be consequence to transcript or protein?
• Allele frequencies across healthy populations and different ethnicities
• Existing evidence in disease
• Plenty of prediction tools for genetic alterations available
Transcriptome
All RNA (transcripts) poduced by cell tissue or organism
Eg mRNA tRNA rRNA
What do we need to make cDNA from RNA
Poly T primers, dNTPS, reverse transcriptase——RNA-DNA hybrid—RNase/ alkali—-ssDNA, dna primers—cDNA
TWO ways to analyse cDNA
- RT-PCR
- .qPCR
How are products detected in qPCR
- SYBR GREEN
- Taq man
SYBR green
Fluroscent molecules bind to double stranded DNA
TAQ MAN
Sequencxe specific probes
CT value
Cycle threshold
The more the dna the quicker the fluroscene will pass the CT value
Used to infer ammount of DNA
GAPDH
Common refrence gene—to infer to cdna
