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I&I SC2 > Molecular diagnostics II > Flashcards

Molecular diagnostics II Flashcards

1
Q

What are the basic characteristics of NGS? (4)

A
  • Millions of reads in parallel
  • Short (<600bp) or long (>10kbp) technology
  • Analysis of complex mixtures of DNA/RNA
  • Enables genome wide approach
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2
Q

Which NGS method can be used for short reads?

A

Illumina

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3
Q

Which NGS methods can be used for long reads? (2)

A
  • PacBio
  • Nanopore
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4
Q

Describe the general workflow for NGS methods

A

Intake -> Isolate -> Library -> Sequence -> Report

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5
Q

What is enrichment and when does this take place in the NGS workflow?

A

Selection of part of the DNA you want to sequence (library step)

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6
Q

What is the definition of a ‘cluster’?

A

Single amplified molecule on a flow cell

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7
Q

What is the definition of a ‘read’?

A

Sequence read from a single cluster

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8
Q

What are the step of DNA/RNA preparation for NGS? (5)

A
  1. Fractionate/size select
  2. End repair/phosphorylate
  3. A-overhang
  4. Add adaptors -> adaptor ligation
  5. Denature and amplify -> product ready
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9
Q

NGS: Fractioning is used for which kind of sequencing?

A

Short-read sequencing

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10
Q

NGS: What does phosphorylation allow for during DNA/RNA preparation?

A

For the addition of adaptors

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11
Q

NGS: What is important to consider when you start from RNA instead of DNA?

A

Reverse-transcriptase step required to convert RNA into ssDNA

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12
Q

NGS: How can you select for non-ribosomal RNA?

A

Selection of fragments that have a poly-A-tail

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13
Q

NGS: What are oligo’s on a flow cell?

A

Anchoring fragments for adaptor molecules attached to the sample fragments

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14
Q

NGS: What does the flow cell do with DNA molecules? How?

A

Amplification -> every cycle, a base is added -> base added is detected

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15
Q

NGS: What is meant with ‘Bridge amplification’?

A

Generation of a cluster around the place where fragments attach to the flow cell

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16
Q

NGS: What does a patterned flow cell allow for?

A

Accumulation of clusters at a determined position

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17
Q

NGS: What are the advantages of a patterned flow cell? (3)

A
  • Reduces overclustering
  • Higher cluster density (data/mm2)
  • No need to map clusters -> reduces runtime
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18
Q

NGS: What is exclusion amplification used by patterned flow cells?

A

As soon as a fragment ends up in a well –> amplification

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19
Q

NGS can/can’t perform multiple reads of a single DNA molecule

A

Can

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20
Q

NGS: What is the advantage of sequencing adaptors?

A

Allows for addition of barcode in adaptor -> distinguish sequences from multiple samples -> simultaneous sequencing

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21
Q

NGS: What does single read/single-end mean?

A

Sequencing a fragment from one side

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22
Q

NGS: What does paired-end sequencing mean?

A

Sequencing a fragment from both sides

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23
Q

NGS: What does a single index mean?

A

One index read (one sample-specific DNA code)

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24
Q

NGS: What does dual index mean?

A

Combined index reads

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25
Q

NGS: What does alignment of detected sequences back to a reference genome to determine its position allow for? (4)

A

Detection of:
- Heterozygous SNPs
- Insertion
- Deletion
- Homozygous SNPs

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26
Q

NGS: What is meant with ‘read depth’?

A

The amount of reads of the sequence that you are interested in

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27
Q

NGS: What is meant with ‘error rate’?

A

Total number mismatched bases after mapping/total number of aligned bases

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28
Q

NGS: What is meant with quality score?

A

Score for the sequence quality per base -> probability that the wrong base was called

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29
Q

NGS: Describe the proces of target enrichment

A
  • Probes(containing magnetic beads)/adaptors are added
  • Magnetic beads are removed
  • Enriched sequences are sequenced
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30
Q

NGS: What is the advantage of enrichment?

A

Deeper sequencing of ROI

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31
Q

NGS: Name applications in which target enrichment is often used (4)

A
  • Exome sequencing
  • Gene panels
  • Low abundant mutations
  • mRNA
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32
Q

NGS: What are approaches to detect viruses using NGS? (3)

A
  • Amplicons -> sequencing part of a sequence using PCR primers
  • Target enrichment
  • Shotgun metagenomics (sequence whole virus)
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33
Q

NGS: What encompasses the technical future of sequencing? (4)

A
  • Amplification-free library prep
  • Longer read length
  • Single cell
  • More combined assays
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34
Q

What is gene expression profiling?

A

Profiles RNA -> presence of genes AND expression levels

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35
Q

Gene expression profiling can be used using? (2)

A
  • Microarrays
  • RNAseq
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36
Q

What are the functions of microarray techniques in AML diagnostics/prognostics? (2)

A
  • Recognize known subgroups
  • Identify novel subgroups
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37
Q

What encompasses the start of any gene expression profiling?

A

RNA isolation

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38
Q

Why is the preparation of intact high-quality RNA essential during gene expression profiling?

A

Critical for obtaining reproducible results

39
Q

Which technique is used to verify the RNA quality and integrity?

A

Automated capillary-electrophoresis

40
Q

Why does the automated capillary-electrophoresis plot alway show two distinct peaks? This is indicative for?

A

18S and 28S ribosomal RNA –> quality of RNA

41
Q

RNA quality depends on…? (2)

A
  • Type: organ, tumor, blood, BM
  • Harvest and storage
42
Q

After quality control, mRNAs are labelled. How?

A

Targeting probes to poly-A-tail

43
Q

mRNA labeling: Which promotor is used when adding a long strand of Ts hybridized to As?

A

Oligo(dT)-T7

44
Q

What is a microarray?

A

Glass slide on which DNA oligo’s are printed

45
Q

Microarray: What do DNA oligo’s printed on the glass slide represent?

A

All genes present in the genome

46
Q

Microarray: What does the intensity of labels (labelled RNA) at certain positions on the array show?

A

Whether and how strongly a gene is expressed

47
Q

Microarrays: what types of arrays are there? (2)

A
  • One colour -> only one sample possible
  • Two colors -> allows inclusion of reference sample
48
Q

What common types of study objectives are used when analyzing micro-array data? (3)

A
  • Class discovery
  • Class comparison
  • Class prediction
49
Q

What is the study objective with class discovery?

A

Are there patterns of cases of disease X with similar expression?

50
Q

What is the study objective with class comparison?

A

Comparing groups to one another -> which genes are specifically up-or down regulated in a particular group

51
Q

What kind of learning is class discovery?

A

Unsupervised -> all data in one bin -> patients with similar expression profile cluster together

52
Q

What kind of learning is class comparison?

A

Supervised -> class labels are added to the samples

53
Q

What is the difference of knowledge taken into account with class discovery/comparison

A
  • Class discovery –> no prior knowledge taken into account
  • Class comparison –> knowledge about certain abnormalities is taken into account
54
Q

Unsupervised learning can be performed using several tools, name 3

A
  • Hierarchical clustering
  • K-means
  • Self-organizing maps
55
Q

Clustering algorithms are based on…?

A

Different assumptions -> correlations between gene expressions

56
Q

The performance of each clustering algorithm depends on..?

A

Properties of the input dataset

57
Q

What are challenges of gene expression profiling in routine diagnostics? (2)

A
  • RNA is highly sensitive to degradation
  • Difficulties in standardizing protocols and techniques
58
Q

Which NGS technique is able to directly sequence mRNA?

A

mRNA-seq

59
Q

mRNA-seq allows for more data, name 5

A
  • Expression levels
  • Fusion transcripts
  • Deletions
  • Insertions
  • Mutations
60
Q

What does the number of reads represent in mRNA-seq?

A

Expression level of certain mRNA

61
Q

What are the advantages of RNA-seq over microarrays? (3)

A
  • Characterize novel transcripts, splicing variants, expression levels of known transcripts
  • Higher resolution
  • Can apply the same experimental protocol to various purposes
62
Q

What are the various purposes of RNA-seq? (3)

A
  • Detecting SNPs
  • Mapping exon junctions (splice variants)
  • Detecting gene fusions
63
Q

If you want to detect SNPs using array technology, which technique do you use?

A

SNP array

64
Q

SNP arrays are used to genotype many variants at the same time. What is the range?

A

Mostly between 750.000-1.2 million SNPs per array

65
Q

Probes attached to one bead are/aren’t for the same SNP

A

Are

66
Q

SNP array: Why is each bead spotted in multifold (multiple times on an array)?

A

To increase accuracy and redundancy

67
Q

SNP array: Describe the procedure (2)

A
  • DNA normalization and whole genome amplification
  • Hybridization on array + single base extension
68
Q

SNP array: how are the SNPs qualified?

A

Green OR red label: homozygous for one variant
Both labels equal: heterozygous

69
Q

SNP array: Why can Illumina not detect CG- or AT SNPs?

A

They use a two-colour system –> C+G and A+T

70
Q

Why can array technology also be used to look for copy number?

A

SNPs are spaced so densely and evenly over the chromosome

71
Q

SNP array: Deletions of … bp can be detected?

A

~90.000

72
Q

SNP array: Duplications of … bp can be detected?

A

~860.000

73
Q

SNP array: Which genotypes are visible when using a range max of 90.000 bp?

A

Only A or B genotypes (No AB)

74
Q

SNP array: What is visible when using a range max of 860.000 bp?

A

Extra band in B-allele frequency (ABB and AAB)

75
Q

SNP array: what is meant with the log R ratio?

A

Normalized measure of signal intensity for each SNP marker

76
Q

In which scenario’s do you apply array technology? (3)

A
  • Rare alleles causing mendalian disease
  • Low-frequency variants with intermediate effect
  • Common variants implicated in common disease by GWA
77
Q

Rare variants are being included in recent arrays, with a special focus on…(3)

A
  • Known pathogenic variants
  • Pharmacogenetic variants
  • Rare, (potentially) pathogenic variants in genes of special interest
78
Q

What is NOT covered by genotyping arrays? (2)

A
  • Non-polymorphic sites of the genome
  • De novo mutations
79
Q

Why are de novo mutations not covered by genotyping arrays?

A

There are too many –> cannot all be covered

80
Q

SNP array: rare variant are/aren’t more difficult to pick up than common variants

A

Are

81
Q

Variants that are unfit to detect using array technology… (2)

A
  • Rare variant with small effect sizes
  • Examples of common variant influencing common disease are rare
82
Q

Why is it advantageous to use arrays-only when having rare alleles with strong effect size?

A

Rare variant directly genotyped on array -> able to screen for all known variants of any given disease as long as they are on the array -> very high accuracy

83
Q

SNP array: Which kind of studies are performed to detect low frequency variants with intermediate effect & common variants implicated in common disease?

A

Genome-wide association studies (GWAS)

84
Q

SNP array: GWAS studies are/aren’t hypothesis free

A

Are -> only assumption = genetic component involved

85
Q

Why is there a huge multiple testing burden in GWAS studies?

A

~1 million independent SNPs for common variants -> big issues with power -> large cohorts needed

86
Q

Describe the steps of a GWAS analysis (3)

A
  • Analyze all SNPs in 1 run
  • Visualizing results in plot
  • Select SNPs with strongest association per chromosome
87
Q

If you found the SNPs with the strongest association per chromosome in a cohort, what is the next crucial step to perform?

A

Replication in a different cohort

88
Q

What are the factors that determine power in GWAS? (3)

A
  • Allele frequency of variant in population
  • Effect size of variant
  • Linkage disequilibrium of variant with true causal variant
89
Q

What is meant with linkage disequilibrium?

A

Chance of inheriting over ‘en bloc’ because they are linked close together -> can seem like a variant is the causal variant, while it only inherits over together with the causal variant

90
Q

Will the causal variant be more present than the LD SNPs around it?

A

Yes

91
Q

What are the applications of GWAS in infectious disease? (with respect to host genetics)

A
  • Infection rate
  • Severity of symptoms
  • Immune response
92
Q

What are the most tested immunological parameters using GWAS?

A

HLA, receptors, etc.

93
Q
A

I&I SC2 (11 decks)

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