Exome

Question 1

NGS is used to identify rare germline variants that cause

Answer 1

rare genetic diseases

Question 2

why do we use NGS

Answer 2

• confirm a clinical diagnosis
• undiagnosed genetic condition
• differential diagnosis
• genetically heterogeneous diseases

Question 3

mendelian disorders are individually - but collectively -

Answer 3

rare, common

Question 4

the 2 main NGS approaches are

Answer 4

• whole genome sequencing

- targeted resequencing (needs the selection of a portion of the genome)

Question 5

targeted resequencing has 2 ways for capture and selection

Answer 5

• amplicon based enrichment

- hybridization based enrichment

Question 6

target sequencing of gene panels is used for -, while of exomes is used for -

Answer 6

• clinical/diagnostic purposes

- research/discovery and clinical use

Question 7

exome

Answer 7

is coding exons and splice junctions, dont contains UTR, are about 38-60 Mb

Question 8

why do we sequence the exome

Answer 8

• it is the part of he genome we understand best
• they are ideal to search for high penetrance allelic variation and its relationship to phenotype
• because variants mutating protein coding sequences and splice sites are more likely to have functional consequences
• less data analysis, less data storage, lower cost

Question 9

what is an advantage of WGS over WES

Answer 9

with WGS you dont miss any info as there is no bias of enrichment, and it detects structural variations better, is a definitive genetic tests

Question 10

what are the steps of a NGS

Answer 10

• study design
• library preparation and sequencing
• data analysis
• data interpretation

Question 11

an overlap based study is used for which cases and what hypothesis

Answer 11

• cases: rare monogenic disorder, recessive/dominant ode of inheritance, unrelated families with same disease/phenotype
• hypothesis: no genetic heterogeneity, shared causative gene, private pathogenic mutation

Question 12

a linkage based study is used for which cases and what hypothesis (DFNB82/GPSM2)

Answer 12

• cases: rare mendelian disorder, low/high genetic heterogeneity, recessive/dominate mode of inheritance, one big family with multiple affected, combine with linage analysis
• hypothesis: shared causative gene and mutation, the gene is likely located in a linkage region

Question 13

a trio design is used for which cases and what hypothesis (mental retardation)

Answer 13

• cases: sporadic patient with non affected parents, monogenic or genetically heterogenous, reduced fertility
• hypothesis: dominant allele, mutation is de novo

Question 14

how is exome data analysis done

Answer 14

• align to the reference genome
• identify variant positions and corresponding genotypes
• annotate variants

Question 15

variant calling

Answer 15

is the process by which nucleotide positions that are different from reference genome are identified and genotype at that position is calculated

Question 16

what makes a good variant calling

Answer 16

• coverage

- appropriate tools are used

Question 17

variant annotation

Answer 17

• quality based: coverage/alignment/genotype confidence (helps in eliminating false positives)
• gene based: localization within gene, effect on protein
• frequency based: presence/absence in public databases (help in selecting functional variants)