Sequencing and resequencinh HG

Question 1

How much of the human genome encodes for proteins?

Answer 1

• about 1.4%

Question 2

Exome capturing

Answer 2

Genomic DNA -> Costruct shotgun library -> Fragments -> Hybridization -> Wash + Pulldown -> Captured DNA -> DNA sequencing -> Mapping, alignment, variant calling

Question 3

Alignment of reads

Answer 3

• align reads on whole reference genome and not just target regions
  
  • reads might not be from target regions but forced to align there
• Reference genome + fq reads -> alignment program + parameters -> result sam

Question 4

Variant callers procedure

Answer 4

• sam file too big to manually analyze
  
  • in lab an exome produces about 40 millions reads
• conversion of sam file into vcf file, each line is a variant
• GATK is the most commonly used program for variant calling
  
  • analyzes NGS data

Question 5

Prioritization in VCF file

Answer 5

• VCF file not easy to analyse without dedicated software
  
  • few genes might be ok but not for large amounts of variants
• variant prioritization takes into consideration many parameters
  
  • quality of the call
  • frequancy of the variant in-population
  • effect of variant
  • biological and medical features

Question 6

Phenotypes and variants

Answer 6

• identify genetic variations that are producing a phenotype
  
  • mutations or polymorphisms
  • set of observable characteristics of an individual
• variant definitions:
  
  • mutation, frequency <1%
  • polymorphism, frequency >1%
• phen caused by a variation in a single gene are easier to study (basic Mendel’s law)
  
  • phen caused by multiple genes are more difficult to characterize

Question 7

Types of variants

Answer 7

• SNP (single nucleotide polymorphism) or SNV (single nucleotide variant)
  
  • generallybiallelic
  • MAF frequency of less abundant one
• Indels
  
  • very short insertions and deletions (one or few bases)
  • less frequent than SNP
• Microsatellites (SSR simple sequence repeats, STR tandem)
  
  • short squences, few bases
  • repeated many times (20)
  • stable within same family
• Structural variations
  
  • large insertions/deletions/inversions
  • can be polymorphic
  • can have detectable phenotypic
• CNV (copy number variations)
  
  • large duplications
  • may be related to diseases

Question 8

Variant analysis, why?

Answer 8

• individual and population characterization
  
  • variant discovery
  • population genetics
  • ancestry
  • genetically driven breeding
• gene hunting
  
  • searching for associated loci
  • linkage analysis
  • searching for causative genes

Question 9

Inheritance of DNA over generations

Answer 9

• 50% from mother and 50% from father
  
  • after n generation we have (1/2^n) DNA from each ancestor
  • 32 generations, one base each
• 2 loci at 1 cMorgan (1% separation by crossing over)
  
  • two loci not separated 0.99
  • n generations 0.99^n
  • in practical we inherit very large regions of genome only from a small number of our ancestors

Question 10

Linkage disequilibrium

Answer 10

• non-random occurrence of alleles at two or more loci in general population
  
  • haplotypes do not occur
• haplotype, group of alleles inherited from a single ancestors
• how much variants are randomly assorted in individuals
  
  • level of linkage disequilibrium is generally high

Question 11

Genome Wide Association Studies (GWAS)

Answer 11

• high level linkage disequilibrium
  
  • genomic sequence = haplotypes + 1/2 mil SNP
  • no new mutations but phenotyper linked to polymorphic variants
• microarrays very useful and cost effective
• allele count of each measured SNP is evaluated
  
  • density usually very high some highly probable
• Manhattan plot helps identifying associated risk loci