Bioninformatics block test 2

Question 1

What is variant calling?

Answer 1

Variant calling refers to how after read alignment we identify positions within a sample that are variable relative to the reference genome used in the allignment.

Question 2

What must be done before variant calling can be carried out?

Answer 2

Duplicates must be removed from the SAM/BAM files.

Question 3

What are the 4 major types of duplicates?

Answer 3

PCR duplicates.
Clustering duplicates.
Optical duplicates.
Sister duplicates.

Question 4

What are PCR duplicates?

Answer 4

PCR duplicates arise due to more than 1 copy of the same fragment annealing to the surface of the flow cell.

Question 5

What are Clustering duplicates?

Answer 5

Clustering duplicates are unique to Illumina and arise during cluster generation when a single library spreads across 2 adjacent tiles on the flow cell.

Question 6

What are optical duplicates?

Answer 6

Optical duplicates also arise during cluster generation, the base caller software reads a single cluster of reads as two, across two adjacent tiles on the flow cell.

Question 7

What are sister duplicates?

Answer 7

Sister duplicates occur when both strands of the same library fragment anneal to the surface of the flow cell.

Question 8

Why is it important to mark and remove duplicates for variant calling?

Answer 8

It is important to remove duplicates for variant calling as duplicates artificially inflate sequencing depth, resulting in homozygous positions being called heterozygous and heterozygous positions being called homozygous.

Question 9

What are the 4 types of variant callers?

Answer 9

1. Copy Number Variant (CNV) callers.
2. Structural Variant (SV) callers.
3. Somatic callers. (somatic mutations)
4. Germline callers (inherited traits)

Question 10

What are the two ways in which variant calling can be preformed?

Answer 10

1. Single sample calling: Per individual.
2. Joint calling: Using information from multiple samples at a time.

Question 11

What is the varient caller pipeline?

Answer 11

Variant caller pipeline:

1. Take SAM/BAM file as input.
2. Mark and remove duplicates.
3. Apply statistical methods to identify variants and assign genotypes.
4. Produce a gVCF file as output.

Question 12

What is variant annotation?

Answer 12

Variant annotation is the process of describing the nature and effect of the DNA alterations produced by a variant.

Question 13

In variant annotation, what does nature and effect refer to?

Answer 13

Nature: Type of sequence alteration (indel, substitution, cnv etc…)

Effect: How the variant changes the annotated reference sequence it occurs in.

Variants will always have a single nature, but can have multiple effects, because the effect on the context of each transcript.

Question 14

Some common varient annotation tools are SnpEff, ANNOVAR, and Varient Effect Predictor (VEP), what do they all have in common?

Answer 14

All varient annotation tools take a VCF file as input, annotate each varient in the file to return an annotated VCF file, with annotations in the info field of the file.

Question 15

What tools are used to predict varient impact on the structure and function of human proteins?

Answer 15

SIFT (Sorting Tolerant From Intolerant), creates a postion-specific weight matrix that corresponds to the profiles of specific protein domains. Position specific weight matrices can also be used to identify transcription factor binding sites.

Question 16

What two factors determine if variation will be retained from generation to generation?

Answer 16

1.) Variation is caused by mutation, if the mutation is in a somatic cell it will not be passed on, but if the mutation occurs in a germline cell it will be.

2. If the mutation positively or negatively affects reproductive fitness.

Question 17

What is the difference between a single nucleotide varient (SNV) and a single nucleotide polymorphism (SNP)?

Answer 17

SNV’s occur in less than 1% of the population.

SNP’s occur in more than 1% of the population.

Question 18

What is the basis of HWE?

Answer 18

In the absence of evolutionary forces, allele, and genotype frequencies remain the same from generation to generation.

Question 19

What are the requirements for HWE?

Answer 19

Large population.
Diploid organisms.
Sexual reproduction.
Allele frequencies equal in both sexes.
Random mating.
No cross generational mating.
No selection.

Question 20

What explains deviations from HWE?

Answer 20

1. Genetic drift
2. Natural selection.
3. Mutation.
4. gene flow.
5. Inbreeding.
6. Non-random mating.

Question 21

What is linkage disequilibrium?

Answer 21

Linkage disequilibrium is the non-random association of alleles at different loci in a given population.

What breaks HWE is what breaks LD as allele frequencies are used to calculate LD.

Question 22

What is a haplotype?

Answer 22

A haplotype refers to alleles that are close together on the chromosome and thus are inherited together in blocks.

Question 23

What are tag SNPs?

Answer 23

Tag SNPs are SNPS that because of Linkage Disequilibrium can be used as markers for specific haplotypes.

Question 24

What is population structure?

Answer 24

Population structure refers to differences in patterns of genetic varients between populations. (with respect to allele frequencies, LD, and haplotype structure.)

Question 25

What is the point of a GWAS?

Answer 25

GWAS identifies correlations between alleles at a particular locus and a phenotypic disease or trait of interest.

Question 26

What is the common disease, common varient hypothesis?

Answer 26

Varients associated with common traits/diseases will have small effect sizes and be common within populations.

Question 27

SNP chips rely on the principle of indirect association, what is that?

Answer 27

The principle of indirect association states that when an association is found between an SNP and a disease, the varient in question is likley no the true varient associated with the disease, but rather one in strong LD wit the responsible varient.

This is becuase tag SNPs are used to represent allelic variability at multiple positions.

Question 28

What is the purpose of diiferential gene expression analysis?

Answer 28

The purpose of differential gene expression analysis is transcriptionally profile and compare 2 conditions, in order to identify genes that are differentially expressed between the two conditions.

Question 29

What are technical replicates?

Answer 29

Technical replicates are repeated measurements of the same sample, they represent measures of random noise associated with the protocols or equipment.

Question 30

What are biological replicates?

Answer 30

Biological replicates are parallel measurements of distinct samples that capture random biological variation.

Question 31

What are eQTLs?

Answer 31

eQTL = Expression Quantitative Trait Loci.

eQTLs: Genomic loci statistically associated with variations in expression.

Question 32

What are local eQTLs?

Answer 32

Local eQTLs are located in close proximity to the genes they influence and can act in cis or in trans.

Question 33

What are distant eQTLs?

Answer 33

Distant eQTLs are located far away from the genes they influence, usually work in trans and have smaller effect sizes, are more tissue-specific, and are harder to detect.