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Genomics > Next Gen Sequencing > Flashcards

Next Gen Sequencing Flashcards

1
Q

When did the human genome project work?

A

1990 - 2003

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2
Q

How many base pairs are in the human genome project?

A

3 billion base pairs long

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3
Q

Which form of sequencing was used in the human genome project?

A

Traditional Sanger Sequencing

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4
Q

How much did the human genome project cost?

A

3 billion dollars

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5
Q

What is PCR and why is it used?

A

It is fundamental for any DNA sequencing application.

PCR is used to amplify a specific region of DNA; primers flank the region to be amplified.

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6
Q

How does PCR work?

A

Each cycle doubles the amount of DNA copies of the target sequence. This amplifes enough DNA molecules so that we have sufficient material to sequence or for other applications.

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7
Q

Briefly explain sanger sequencing

A
  • Invented by Fred Sanger in 1977.
  • Cycle sequencing
  • One reaction = one sequence
  • Accurate (99.99%)
  • Slow and low throughput
  • Used predominantly until late 2000s
  • Costly
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8
Q

Why is Next Gen sequencing a preferred way of sequencing?

A
  • It matches the technological advances since the end of the human genome project.
  • Decrease in the cost of DNA sequencing
  • Since the end of 2007, the cost has dropped at a rate faster than that of Moore’s law
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9
Q

What is Next Generation Sequencing used for?

A
  • It has replaced Sanger Sequencing for almost all sequencing tests in the lab
  • Whole genome sequencing
  • Whole exome sequencing
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10
Q

What are the four steps in next gen sequencing?

A
  1. DNA library construction
  2. Cluster generation
  3. Sequencing-by-synthesis
  4. Data analysis
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11
Q

What is step 1 - DNA library construction?

A
  • In the wet lab, prepare the DNA sample for sequencing
  • DNA is chopped into small fragments (typically 300bp). This is called shearing
  • This can be achieved chemically, enzymatically or physically (sonication).
  • Repair the end of the sheared DNA fragments by adding adenine (A) nucleotide overhangs
  • Adapters with thymine overhangs can be ligated to the DNA fragments
  • End result is the DNA library of literally billions of small, stable random fragments representative of our original DNA sample
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12
Q

What is shearing?

A

The process of chopping DNA into smaller fragments by chemicals, enzymes or physical process (sonication).

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13
Q

What is a DNA library?

A

A collection of random DNA fragments of a specific sample to be used for further study; for example, next gen sequencing.

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14
Q

Why are adapters important in step one of DNA library construction?

A
  • Adapters contain the essential components to allow the library fragments to be sequenced
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15
Q

Give examples of adapters added to the sequence

A
  • Sequencing primer binding sites

- P5 and P7 anchors for attachment of library fragments to the flow cell

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16
Q

What is step 2 - cluster generation?

A
  • Hybridise DNA library fragments to the flow cell. This is a random process.
  • This is needed to amplify the fragments to a bigger size that we can measure as a lot of it in the DNA library is too small.
  • Perform bridge amplification to generate clusters
  • Clusters are now big enough to be visualised and the flow cell is ready to be loaded onto the sequencing platform
17
Q

What is step 3 - sequencing by synthesis?

A
  • Modified 4 bases (ATCG) with chain terminators.
  • Different fluorescent colour dye so each single nucleotide is sequenced 1 cycle at a time in a controlled manner.
  • Single nucleotide incorporation (DNA polymerase); flowcell wash. Image the 4 bases (digital photograph).
  • Cleave termination chemical group and dye with enzyme
  • Camera sequentially images all 4 bases on the surface of the flow cell each cycle.
  • Each cycle image is converted to the nucleotide base call (ACGT).
  • Cycle number is anywhere between 50-600 nucleotide base pairs.
18
Q

Compare the NGS vs sanger sequencing

A
  • NGS produces a digital readout whereas sanger sequencing produces an analogue readout.
  • Sanger is one sequence read whereas NGS is a consensus sequence of many reads
19
Q

Why is exome sequencing preferred to genome sequencing?

A
  • Only interested in the gene protein coding exons or ‘exome’ represents 1-2% of the genome.
  • It is more efficient to only sequence these parts rather than the whole genome as it costs £1000 to do the entire genome, but only £200-300 for an exome.
  • 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
20
Q

What percentage of pathogenic mutations are protein coding?

A

About 80%

21
Q

What are the different exome data analysis techniques?

A
  • Sequence Read Alignment
  • Target Coverage Reporting
  • Variant Annotation
  • Variant Calling
22
Q

Why is exome sequencing done?

A
  • To collect disease affected individuals and their families
  • Use of NGS in disease gene identification
  • Perform exome sequencing
  • Compare variant profiles of affected individuals
23
Q

What is being looked for in exome sequencing?

A

The mutations in the protein coding in the exons

24
Q

NGS is not just for DNA, What else can it be used for?

A

RNA sequencing using the total RNA (or mRNA) from a collection of cells or tissues.

25
Q

How does RNA-seq using NGS work?

A
  • RNA is first converted to cDNA prior to library construction.
  • NGS of RNA samples determine which genes are actively expressed
  • Single experiment can capture the expression levels of thousands of genes
26
Q

What is used as a measure of gene abundance?

A

The number of sequencing reads produced from each gene.

27
Q

Why is RNA-seq carried out?

A

To discover distinct isoforms of genes that are differentially regulated and expressed

28
Q

What is third-generation sequencing? Give an example

A
  • Single molecule sequencing

- An example is Oxford Nanopore sequencing.

29
Q

What is oxford nanopore sequencing?

A

This is where DNA passes through a nanopore and base sequence is converted into an electrical current

30
Q

Advantages of oxford nanopore sequencing

A
  1. No expensive machine required
  2. Each flowcell is the machine itself
  3. Scalable to required throughput
31
Q

Disadvantages of oxford nanopore sequencing

A
  1. Very expensive
  2. High error rates
  3. Technology is still developing

Genomics (19 decks)

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