Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

SGUL - Genomics > Next Generation Sequencing > Flashcards

Next Generation Sequencing Flashcards

1
Q

Briefly describe the Polymerase Chain Reaction

A
  • Fundamental principle for any DNA sequencing application
  • Used to amplify a specific region of DNA, Primers flank the region you want to amplify
  • Each cycle doubles the amount of DNA copies of your target sequence
  • Amplify enough DNA molecules so that we have sufficient material
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Briefly describe Sanger sequencing

A
  • Invented by Fred Sanger in 1977
  • Cycle sequencing
  • Based on PCR (also needs a PCR product as an input)
  • Needs primers
  • Modified nucleotides: Chain terminators or nucleotide specific colour tag
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What does Sanger sequencing identify?

A
  • A single nucleotide polymorphism (SNPs) or mutations
  • Can identify monogenic disease causing mutations
  • Often used for single gene tests
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are the benefits of next generation DNA sequencing?

A
  • Decrease in the cost of DNA sequencing
  • Since the end of 2007, the cost has dropped at a faster rate than that of Moore’s law
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

When was Next generation of DNA sequencing developed?

A
  • Development of NGS methods began 13 years ago with 454 pyrosequencing
  • DNA sequencing throughput jumped 10 orders of magnitude
  • Solexa sequencing by synthesis (SBS) developed end of 2005
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What happened as a result of the development of next generation sequencing?

A
  • Replaced Sanger sequencing for almost all sequencing in the lab
  • Whole genome sequencing
  • Whole Exome sequencing
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are the four steps of NGS?

A
  1. DNA library construction
  2. Cluster generation
  3. Sequencing by synthesis
  4. Data analysis
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Describe the steps in DNA library construction (PART 1)

A
  • Takes place in the wet lab
  • DNA is first chopped into small fragments (typically 300 bp). called shearing
  • Can be achieved chemically, enzymatically or physically (sonication)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Describe the steps in DNA library construction (PART 2)

A
  • The end of the sheared DNA fragments have to be repaired
  • Adenine nucleotide overhangs are added to end of fragments
  • Adapters with Thymine overhangs can be ligated to the DNA fragments
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Describe the steps in DNA library construction (PART 3)

A
  • Adapters contain the special components to allow the library fragments to be sequenced
  • Sequencing primer binding sites
  • P5 and P7 anchors for attachment of library fragments to the flow cell
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Describe the steps in cluster generation (PART 1)

A
  • Hybridise DNA library fragments to flowcell
  • But we can’t visualise individual single molecules of our DNA library: too small
  • We need to amplify the fragments to a bigger size for a stronger signal
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Describe the steps in cluster generation (PART 2)

A
  • Perform bridge amplification to generate clusters
  • Many billions of clusters originating from single DNA library molecules
  • Clusters are now big enough to be visualised
  • Flow cell is now ready to be loaded
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Describe the steps in Sequencing By Synthesis (PART 1)

A

Modified 4 bases (ATCG) with:
- Chain terminators
- Different fluorescent colour dye

  • Sequence each single nucleotide 1 cycle at a time in a controlled manner
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Describe the steps in Sequencing By Synthesis (PART 2)

A
  • Single Nucleotide incorporation (DNA polymerase)
  • Flowcell wash
  • Image the 4 bases (digital photograph)
  • Cleave terminator chemical group and dye with enzyme
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Describe the steps in Sequencing By Synthesis (PART 3)

A
  • Camera sequentially images all 4 bases on the surface of the flowcell each cycle
  • Each cycle image is converted to a nucleotide base cell
  • Cycle number is anywhere between 50 - 600 nucleotide base pairs
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Describe the last stage: Analysis of NGS data

A
  • Short read sequences from the sequencing machine need to be pieced together like a jigsaw
  • Mapping locations of our sequence reads on the reference genome sequence
  • To generate a consensus sequence of original DNA sample library
17
Q

Compare and contrast between NGS and Sanger Sequencing

A
  • NGS produces a digital readout.
  • Sanger produces an analogue readout
  • Sanger is one sequence read
  • NGS is a consensus sequence of many reads
18
Q

How many genes are there in the human genome?

A

21,000 Genes

19
Q

What part of the gene are we usually interested in?

A

In the gene protein coding exons or ‘Exome’ which represents 1-2% of the genome

20
Q

What percentage does pathogenic mutations acquaint for?

A

80% of pathogenic mutations are protein coding

21
Q

What can we do with the whole exome sequencing to be more efficient?

A

More efficient to only sequence the bits we are interested in rather than the whole Exome sequencing

22
Q

What is Whole Exome Sequencing used for?

A
  • Target enrichment
  • Capture target regions of interest with baits
  • Potential to capture several Mb genomic regions of interest
  • Exome would be 50 Mb in size
23
Q

How is Exome Data Analysis carried out?

A

Sequence read alignment -> Target Coverage Reporting -> ? -> Variant Annotation

24
Q

How is Whole Exome Sequencing used?

A
  • The target is to look for protein coding mutations in the exon
  • The patient DNA sample is subjected to Exome sequencing
  • This should show a snippet of the consensus sequence of the sequence sample
  • this reveals a heterozygous mutation in the CFTR gene
25
Q

What are the applications of Exome Sequencing?

A
  • Collecting disease affected individuals and their families
  • Use of NGS in disease gene identification
  • Perform Exome sequencing
  • Compare variant profiles of affected individuals
26
Q

What is RNA sequencing 1?

A

RNA-seq experiments use the total RNA (or mRNA) from a collection of cells or tissue

27
Q

What occurs in RNA-sequencing?

A
  • RNA is converted to cDNA before library construction
  • NGS of RNA samples are used to determine which gene are actively expressed
  • Single experiment can capture the expression levels of thousands of genes
28
Q

What occurs in RNA-sequencing? (PART 2)

A
  • The number of sequencing reads produced from each gene can be used as a measure of gene abundance
  • Quantification of the expression levels
  • Calculation of the differences in gene expression of all genes in the experimental conditions

SGUL - Genomics (21 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions