Question 1

What are the four steps in Next Generation Sequencing

Accepted Answer

Sample Prep Cluster Generation Sequencing Data Analysis

Question 2

What is involved in Sample Preparation

Accepted Answer

Fragmentation Adaptor ligation

Question 3

What do the adaptors consist of

Accepted Answer

Sequencing binding sites Indices Regions complementary to the flow cell oligos

Question 4

What are the steps involved in cluster generation

Accepted Answer

Hybridization of DNA to flow cell oligos Polymerase generates a complementary strand Denaturation Clonal/Bridge amplification Reverse strand cleavage 3' blocking

Question 5

What is involved in Sequencing

Accepted Answer

Sequencing by synthesis which is extension of sequence primer with fluorescent blocked dNTPS (one addition at a time)
Light excitation of cluster emits colour based on specific nucleotide

Massively parallel sequencing

Indices are for multiplexing (mutiple samples)

Reverse strand is also sequenced, generating pair end reads of 36bp on each end of the fragment

Question 6

What is the steps of data analysis

Accepted Answer

Samples can be separated based on index seqeunces Sequences are aligned to a reference Paired ends allow resoultion of ambiguous alignments

Question 7

What is the application whole genome sequencing

Accepted Answer

Determines the complete DNA sequence of an organisms genome at a single time by overlapping reads which creates a library

Look for genetic aberrations (SNVs. deletions,insertions, CNVs) in coding and non-coding regions
Example: Identify causative variants associated with complex disease and traits
De novo sequencing

Question 8

What is application of whole exome sequencing

Accepted Answer

Sequencing of all protien-coding genes in the genome by selecting only the subset of DNA that encodes protiens
Exons + splice junctions and 150nt into the introns

Example: Search for mutations causing Mendelian disorders
Example: Search for gene variants associated with complex genetic diseases

Question 9

What is the application of RNA-seq

Accepted Answer

Measures the levels of mRNA molecules expressed from the genes of an organisms the transcriptome

Example: Studying expression levels associated with a certain disease or how the expression leveles react to treatment

Detect inherited and de novo mutations
Quantify mutations burden and levels of mosaicism
Mutations due to RNA editing

Question 10

What is the application of target gene/exome sequencing

Accepted Answer

A select number of genes or coding regions within genes of intrest are selected for and sequenced
Genes are known to harbour mutations that contribute to the pathogenesi of a disease
Example: targeted disease disease panels focus on select gens or gene reigions that have known associations with the disease (Skin Cancer0

Question 11

What are advantages of WGS

Accepted Answer

Highest coverage Captures both large small variants

Question 12

What is limitations of WGS

Accepted Answer

More expensive Time-commitment Complex bioinformatics pipeline

Question 13

What are advatnages of WES

Accepted Answer

Less expensive Greater depth less bioinformatics work

Question 14

What are limitations of WES

Accepted Answer

Won't see variants in non-coding regions

Question 15

What are advantages RNA-Seq

Accepted Answer

Less expensive Allows studies of gene expression Less bioinformatics work

Genomic Methods 2 Flashcards

(27 cards)