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Genomics > Next Generation Sequencing > Flashcards

Next Generation Sequencing Flashcards

1
Q

When was the human genome project completed?

A

Human Genome Project (1990 - 2003) costing $3bn

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2
Q

How base pairs long is the human genome project?

A

3 billion base pairs long

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3
Q

What technique was used to produce the human genome?

A

All done with traditional Sanger Sequencing

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4
Q

What is the significance of the human genome project?

A

Unravelled the first Human Genome Sequence to drive genetics research
We can now achieve this amount of sequencing in as little time as one day!

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5
Q

What is the use of PCR?

A

PCR is used to amplify a specific region of DNA; primers flank the region you want to amplify
- Amplify enough DNA molecules so that we have sufficient material to sequence or for other applications

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6
Q

Why is PCR so significant?

A

Fundamental for any DNA sequencing application

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7
Q

Why is PCR so great at amplifying DNA?

A

Each cycle doubles the amount of DNA copies of your target sequence

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8
Q

What is Sanger Sequencing?

A

Invented by Fred Sanger back in 1977
Cycle Sequencing; based on the same principles as PCR
Each of the 4 DNA nucleotides has a different dye so we can determine the nucleotide sequence

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9
Q

Outline the advantages of Sanger Sequencing

A
  • Up to 800 bp of sequence per reaction
  • Accurate (99.99%)
  • One reaction = one sequence
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10
Q

Outline the disadvantages of Sanger sequencing

A
  • Slow and low-throughput

- Costly to perform ££££

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11
Q

What are the benefits of next generation sequencing?

A

Technological advances since the end of the human genome project
Decrease in the cost of DNA sequencing: since2007, the cost has dropped at a rate faster than that of Moore’s law

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12
Q

Describe the 4 key principles of NGS

A

DNA library Construction
Cluster Generation
Sequencing-by-synthesis
Data analysis

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13
Q

What is a DNA library?

A

A DNA library is a collection of random DNA fragments of a specific sample to be used for further study; in our case next generation sequencing

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14
Q

Where do we obtain DNA from for DNA libraries?

A

The DNA can come from just about anywhere, but in human genetic research generally it’s derived from patients blood.

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15
Q

How is DNA prepared for a DNA library construction?

A

In the wet lab DNA prepped for sequencing
DNA chopped into small 300 bp fragments
=> Shearing

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16
Q

How is shearing achieved?

A

Shearing achieved chemically, enzymatically or physically (sonication)

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17
Q

How are the ends of the sheared DNA repaired?

A

Adenine (A) nucleotide overhangs are added to the end of fragments
Adapters with Thymine (T) overhangs can be ligated to the DNA fragments

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18
Q

Describe the products of DNA library construction in NGS

A

The end result is the DNA library of literally billions of small, stable random fragments representative of our original DNA sample

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19
Q

What are adaptors used for in NGS DNA library constructions?

A

Adapters contain the essential components to allow the library fragments to be sequenced

20
Q

What is the purpose of P5 and P7 anchors?

A

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

21
Q

How does cluster generation occur?

A

Hybridise DNA library fragments to the flowcell (microscope slides sandwiched together)
This is a random process

22
Q

Why do we carry out PCR during cluster generation?

A

We can’t see individual single molecules of DNA library –too small
We use PCR to amplify the fragments to a visible size

23
Q

How are clusters generated during NGS?

A

Bridge PCR generates clusters originating from the single DNA library molecules
Clusters are now big enough to be visualised

24
Q

How is sequencing of the clusters by sequencing carried out?

A

Sequence each nucleotide 1 cycle at a time in a controlled manner
Modified 4 bases (ATCG) with chain terminators AND a different fluorescent colour dye

25
Q

Explain how sequencing by synthesis occurs?

A
  1. Single nucleotide incorporation (DNA polymerase)
  2. Flowcell wash
  3. Image the 4 bases (digital photograph)
  4. Cleave terminator chemical group and dye with enzyme
  • repeat n times for a full length sequence
26
Q

How is the sequencing by synthesis presented?

A

Camera sequentially images all 4 bases on the surface of the flowcell each cycle

27
Q

What can we determine from each cycle image of the flowcell?

A

Each cycle image is converted to a nucleotide base call (ACGT)

28
Q

How many cycles are there for each flow cell?

A

Cycle number anywhere between 50 – 250 nucleotide base pairs

29
Q

How do we analyse short read sequences produced via NGS?

A

To generate a consensus sequence of our original DNA samples short read sequences from the machine need to be re-assembled like a jigsaw

30
Q

How can we use the consensus sequence to identify genetic variants?

A

We can compare this consensus sequence against the human genome reference and look for the genetic variants

31
Q

What tools are used to compare consensus sequences to the human genome?

A

Dedicated software and bioinformatics tools will achieve this

32
Q

Approximately how many genes are there in the human genome?

A

There are ~21,000 genes in the human genome

33
Q

Of the human genome, which areas are most interesting to study for variants?

A

Often we are only interested in the gene protein coding exons or ‘exome’ represents 1-2% of the genome

34
Q

How many pathogenic mutations are found in exomes?

A

Some ~80% pathogenic mutations are protein coding

35
Q

What is the benefit of only looking at the exome?

A

More efficient to only sequence the bits we are interested in, rather than the entire genome
Costs £1,000 for a genome, but only £200-£300 for an exome

36
Q

How is exome sequencing achieved using target enrichment?

A
  1. Samples + buffer + baits
  2. Hybridisation occurs
  3. Add coated magnetic beads
  4. Capture beads bound to samples
  5. Unbound traction discarded
  6. Wash beads and digest RNA
  7. Amplify for sequencing
37
Q

Why is target enrichment a good method used to prep samples for amplification?

A

Potential to capture several Mb genomic regions of interest

Exome would be 50Mb

38
Q

How does NGS differ from Sanger sequencing?

A

NGS

  • produces a digital readout.
  • consensus sequence of many reads

Sanger

  • produces an analogue readout
  • one sequence read
39
Q

How can genetic diseases be explored using NGS?

A

Collecting disease affected individuals and their families
Perform exome sequencing
Incorporation of NGS into disease gene identification

40
Q

Where is RNA obtained for RNA sequencing using NGS?

A

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

41
Q

Outline how NGS is used for RNA sequencing

A
  1. RNA is first converted to cDNA prior to library
    construction
  2. NGS of RNA samples determine which genes are
    `actively expressed
42
Q

How can we determine gene abundance from NGS RNA sequencing?

A

The number of sequencing reads produced from each gene can be used as a measure of gene abundance

43
Q

How is NGS RNA Sequencing able to help us identify differing gene expressions

A

With appropriate analysis, RNA-seq can be used to discover distinct isoforms of genes are differentially regulated and expressed

44
Q

What methods are used in third-generation sequencing?

A

Oxford Nanopore sequencing
Single-molecule sequencing
No PCR involved

45
Q

How is third-generation sequencing carried out?

A

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

Genomics (20 decks)

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