!Lecture Objectives - Be able to cite examples of genetic variations!- Understand the distinction in: targeted DNA sequencing, whole exome sequencing, and whole genome sequencing!- For each sequencing approach understand: uses, advantages, and limitations!- Be able to describe criteria for DNA sequence: analytical validity, clinical validity!!Human Genome Project!- In the past 20 years, the rate of DNA seq. has increased a million fold, cost decreasing w/that!- Will entire genome become part of each person's med record--personalized medicine?!- The entire 3x10^9 bp sequence is a "representative genome" coding for about 20,000 genes!- about 2% codes for proteins!- Lots of repetitive DNA!- You and person next to you share about 99.9% sequence identity (=a million differences)!- Variation occurs about 1/300 bp (SNP) in a popn!- SNP= single nucleotide polymorphisms, copy number variations!-->associated w/disease risk, outcome, response!- a single DNA base variation can impact many diff. things-ex. change aa, DNA reg. seq., RNA splicing...etc!- Genotype= phenotype= individually unique!- Other genetic variations assoc. w/disease: copy # variations, deletions, insertions, epigenetic, chrom. translocation, aneuploidy!- Single gene variations that cause disease!- Ex. Huntingtons. Easy to diagnose bcause when that gene is mutated in certain ways-->cause disease - Single gene variations that increase risk - genes that are assoc. w/a certain disease when they're mutated!- Ex. Brca1/breast cancer-gene mut. itself didn't cause cancer-->increased risk-->cancer - Multi-genetic variations that influence health and disease - lots of genes contribute to a particular gene, multiple pathways, more difficult and time consuming !- Ex. Cancer, obesity, Diabetes, etc.!!Approaches to Analyze a genome!- Cytogenetics (karyotypes)!- Can provide diagnostic info on well est. chrom. abnormalities ex. Down Syndrome and identify new structural variations potentially!- Can't identify small structural variations or single base changes!- DNA sequencing methods!1) Fragment the DNA!2) Sequence small segments!3) Reassemble sequence reads!- Computer aligns all the small seqs where they are contiguous/overlapping. Now, because the genome has been seq., you can align them against the "reference genome" (a popn representative) --> easier to realign all the seqs!- Targeted gene sequencing - sanger chain termination method= dideoxy sequencing (takes care of the below wants)!- Sanger sequencing reads fragments from different alleles (picks up that C you got from yourmom and the T you got from your dad)!1) DNA preparation, target amplification or target selection!2) Gene specific primer directed chain extension - Primers provide specificity - DNA polymerase builds chains base by base!- Primers increase sensitivity (becuasae PCR boosts the target copies!)!3) Dideoxy chain termination!- tagged (fluor.) bases randomly terminate chains!4) Capillary electrophoresis!- fragments sort by size and detector reads each tagged base!- DNA is - charged so it migrates toward + electrons--small fragments move faster than large fragments!5) Computer analysis!- Want:!- Specificity (for the target)= picking the right target to seq. - Sensitivity to pick up single base variations - Analytical validity - accurate and reliable - Clinical validity- useful - Illumina Next Gen Seq- Bridge Amplification!- Bridge amplification increases sensitivity!- Immobilize primers on flow cells--> we have sensitivity and specificity!- DNA fragments are immobilized in clusters, each cluster contains a different starting fragment of DNA!- PCR rxn still occurs!- Primers being used are NOT part of the target genomic seq.!- Target amplifies in cluster!- Fluor. bases are added one a time!- Digital camera detects each base addition in real time!- Key concept: NexGen seq. is so powerful because the flow cell can have 1000s to millions of fragments of DNA being seq. at the SAME TIME, and the high resolution of the digital camera can monitor each one. --> doesn't take long!!- quality control- want 30 reads/every seq.!- computer aligns all the seq. fragments to get a full seq.!- the contiguous seq. can be the entire genome!!- Analytical validity= measure of accuracy of seq. read, most labs use sanger to verify - Clinical validity= which variants are causal? still need validation databases of variant all fluor. bases 1 at a timeassociation w/disease trait. - Exome sequencing - only the 2% of genome of protein coding DNA!- isolate the mRNA from the target cell-->RNA bait in test tube w/genomic DNA-->RNA bait hybridizes to target DNA seq.-->select only the hybrids for sequencing!- Pros: more limited data, higher coverage, easier to assemble, costs less, targets all proteins!- Cons: misses variations in introns, regulatory regions, non-coding RNAs, may still have sequencing gaps - Whole genome sequencing - all 6 billion bases in a genome!- Pros: includes all sequences= more comprehensive - Cons: more expensive to run and analyze, more prone to erros, some areas of DNA don't seq. well, may identify more variants than you know what to do with, variant of undetermined significane (VUS), incidental findings (find things unrelated to what you were looking for)!- somatic (inherited) genome!- disease tissue (e.g. cancer) look for
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