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Genomics > Introduction to sequencing > Flashcards

Introduction to sequencing Flashcards

1
Q

Describe the order of events that take place when carrying out genetic analysis

A

Patient sample is take

DNA is prepared

Sequencing

Data analysis

Data integration and preparation

Identificaiton of biomarkers

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2
Q

What is the first step that needs to be taken to sequence a material?

A

It needs to be prepared

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3
Q

How is a sample prepared for sequencing?

A

FFPE

Formalin fixed, paraffine embedded

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4
Q

Describe the process of FFPE

A

Surgical or core needle biopsies are taken

Take samples, fix them with formalin and embed them in paraffin

Take a slide and form a histological analysis

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5
Q

Why are liquid biopsies more difficult to handle?

A

Contain free floating DNA, so it is more easily degraded

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6
Q

What is another way to obtain DNA for sequencing, apart from FFPE?

A

Short fragments of DNA found in exosomes can be removed using surfactants and RNAses to degrade the surrounding phospholipid bilayer

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7
Q

What information can be obtained from sequencing and microarrays?

A

Genomics

Transcriptomics

Epigenomics

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8
Q

What is epigenomics?

A

Changes that affect proteins expression (which and at what level) without affecting the underlying DNA sequence

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9
Q

What are the main types of epigenetic modifications?

A

Methylation

Acetylation

Histone modification - looked at using different procedures

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10
Q

What is the difference between sequencing and microarrays?

A

Both can be used to read the sequence of DNA, RNA or epigenetic modifications

But sequencing measures the full sequence or section of DNA, whereas microarrays measure large numbers of genetic variants simultaneously

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11
Q

Explain the composition of microarrays

A

Chips of glass (8cm x 2cm)

With many oligonucleotides bound to them

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12
Q

What are oligonucleotides?

A

Microscopic spots on solid surfaces

Specific, short DNA sequences

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13
Q

What is the function of oligonucleotides?

A

Hybridise to specific DNA seqences to tell where variants in the genome are

Oligos are specific to variants you want to identify

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14
Q

How must a sample be prepared in order to carry out sequencing?

A

The sample must be fragmented

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15
Q

Two methods of fragmentation used for preparation of sequencing material

A

Sonication

Enzymatic fragmentation

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16
Q

What is sonication?

A

The use of sound to split the DNA into fragments

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17
Q

What is enzymatic fragmentation?

A

The use of enzymes to fragment the DNA in a predictable manner

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18
Q

What is the main difference between fragmentation using sonication vs enzymatic fragmentation?

A

Enzymatic fragmentation is predictable, whereas sonication is random

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19
Q

What is a way to ensure all of the DNA is sequenced?

A

Sonication can be used repeatedly on the same molecule to obtain different variations of the fragments

This eliminates the possibility of missing part of the genome

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20
Q

What is a disadvantage of enzymatic degradation?

A

Often lead to fragments missing gaps of the genome

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21
Q

What is done to the DNA after fragmentation?

A
  1. Repair the ends of the fragments using high fidelity polymerase
  2. Attachment of adapters to the sequences via ligation
  3. Sequencing
  4. Align to the genome
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22
Q

What does the polymerase have to ensure when fixing the fragmented strands?

A

Make sure the 3’ hydroxyl group and the 5’ phosphate group are present on either end

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23
Q

What is important regarding the adapters that are ligated to the fragmented sequences?

A

Specific to the technology used to read the sequence

Needs to bind to probes with complementary strands

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24
Q

What must be done in order to only sequence the exonic sequences of DNA?

A

Use special RNA tags containing magnetic beads

These tags are specific to the 100-120 bases of the exonic sequences

A magnet is applied to extract the exonic bases

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25
Q

Why are the special magnetic RNA tags so long?

A

To ensure that even the variants of the genes are also extracted

If the sequence is not perfect because of it being a variant, the bonds with the rest of the DNA strand that is complementary will compensate

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26
Q

Advantages of sequencing over microarrays

A

Greater coverage across the whole genome

Greater versatility

Can identify novel gene variants and structural variations

Unbiased

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27
Q

Disadvantages of sequencing over microarrays

A

Expensive

Very complex analysis

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28
Q

Advantages of microarrays over sequencing

A

Cheap

Well validated analysis method

Relatively low amount of inpit DNA

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29
Q

Disadvantages of microarrays over sequencing

A

Limited to a certain number of known loci

Depends on prior sequence knowledge

Biased

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30
Q

What does RNA sequencing look at?

A

Differences in transcriptome between abnormal and normal cells

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31
Q

What information does RNA sequencing provide?

A

Shows which genes are active and how much they are transcribed

32
Q

Describe the steps of RNA sequencing

A
  1. Prepare RNA-seq library
  2. RNA and mRNA is obtained (with DNA = contamination)
  3. Convert RNA into DNA
  4. Add sequencing adaptors
  5. Amplify using PCR (only those with adaptors will amplify)
  6. Sequence
33
Q

What is done when the two samples of RNA are sequenced?

A

Compare the sequences between normal and mutated cells to identify the underlying mutation

34
Q

Why are the RNA sequences transformed into DNA?

A

More stable

Can be more easily modified

35
Q

What is the use of sequencing adaptors in RNA sequencing?

A

Allows the sequencing machine to recognise the fragments

Allows scientists to sequence different samples at the same time through using different adaptors

Only the fragments with adaptors will be amplified in the PCR machine

36
Q

What is important to do before sequencing a prepared sample?

A

Quality control

  • verify library concentration
  • verify library fragment lengths
37
Q

Why are not all the RNA fragments sequenced?

A

Since certain parts of the transcriptome are not informative

38
Q

What does methylation do?

A

Silence the transcription of a certain gene

39
Q

What technique is the gold standard for looking at the methylation of a DNA sample?

A

Bisulfite conversion

40
Q

Describe the steps of bisulfite conversion

A
  1. Bisulfite targets the unmethylated cytosines, converting cytosine into uracils through deamination
  2. The uracils are converted into Ts
41
Q

How is bisulfite conversion used to look at methylation?

A

You run a bisulfite conversion and a next generation sequence on the same sample

Compare the position of cytosines and thymines

If a C in the next generation sequence is converted into a T in the bisulfite conversion sequence, it is an unmethylated site

42
Q

Which areas of the genome does methylation occur?

A

CpG sites

Where a cytosine is found next to a guanine

43
Q

Why is looking at methylation patterns in DNA so expensive?

A

Need to sequence the DNA twice

Through next generation sequencing and bisulfite conversion

44
Q

What is the original sequencing technique called?

A

Sanger sequencing

45
Q

Is Sanger sequencing still being used?

A

Yes

Because of its low cost

46
Q

Describe how a Sanger sequence is carried out

A
  1. ddNTPs are added to the mixture containing single strands of the DNA you want to analyse
  2. These are labelled using a fluorescently coloured tag
  3. Once incorporated through complementary base pairing, replication ends due to the nature of the ddNTPs
  4. The different fragments are then separated by their weight using gel electrophoresis - allowing scientists to determine the position of nucleotides
47
Q

Why is so much DNA input required for Sanger sequencing?

A

The procedure is based on chain termination

So we need to read the same molecular at different times along the molecule 1

48
Q

What about the nature of ddNTPs allows chain termination to take place?

A

Lack a hydroxyl group, so cannot form a phosphodiester bond with the next complementary nucleotide

49
Q

What is the current gold standard for sequencing?

A

Illumina sequencing

50
Q

Describe the three main steps of Illumina sequencing

A

Library preparation

Cluster generation

Sequencing

51
Q

Describe the steps of library preparation

A

Flow cells contain complementary sequences to the adaptors bound to the DNA formed through sample preparation

The adaptor on one end binds to the complementary sequence on the probes

Polymerase makes a new strand vertically, which is bound to the flow cell using another oligo probe

The old strand is washed away

52
Q

Describe the process of cluster generation

A

The new strand bends over to hybridise the other oligo complementary to the sequence on the other side of the strand

This is repeated many times to form a cluster

Once all the single strands are formed, the reverse strands are washed away and the 3’ ends are blocked to prevent unwanted priming

53
Q

Describe the process of illumina sequencing

A

A sequencing primer is added together with fluorescently labelled nucleotides

The nucleotides add on to the growing chain

Every time a bond is formed between complementary nucleotides, a fluorescent signal is emitted depending on the nucleotide added

A picture is taken every time the colour is emitted

54
Q

What determines the base added to the sequence?

A

The colour of the bed

55
Q

What determines the length of the read?

A

The number of pictures taken

56
Q

What is the purpose of the clusters?

A

The radioactive signal emitted is very small

The generation of clusters of the same sequence of DNA means that the signal is amplified, making it easier for the bases to be determined more accurately

57
Q

Why are the same sequences read forwards and backwards?

A

Because the fact that the signals detected rely on simultaneous emission of the light, means that fidelity decreases with time

The light emitted becomes weaker as the signals formed are less synchronised

58
Q

Where does most of the cost of Illumina go?

A

To storing the images

59
Q

What is the average cost of Illumina sequencing?

A

£5500 per run

60
Q

What are examples of sequencing by synthesis?

A

Pyrosequencing

Ion torrent

61
Q

What does pyrosequencing detect?

A

Light emitted following the formation of a bond between two bases

62
Q

Describe the process of pyrosequencing

A
  1. For every nucleotide incorporated, a molecule of pyrophosphate is release
  2. The pyrophosphate is converted into ATP through an ezymatic reaction using ATP sulfurylase
  3. ATP converts luciferin to luciferase, which emits light
63
Q

What information does the light frequency provide us?

A

Number of bases in a row

Light frequency generated determines the base added

64
Q

What does ion torrent detect?

A

pH changes as H+ are released upon the formation of phosphodiester bonds

65
Q

Describe the process of ion torrent

A
  1. Phosphodiester bond is made, releasing H+
  2. A pH meter will detect the change in pH, informing the analyst which base has been added since the change in [H+] varies depending on the base added
66
Q

What information does ion torrent provide?

A

The base added

67
Q

What is a disadvantage of ion torrent?

A

Not too accurate

The difference between 1H+ and 2H+ is so small that it is often not picked up

68
Q

What is the future of sequencing?

A

3rd generation technologies

69
Q

Example of a third generation sequencing technology

A

Nanopore

70
Q

What is a nanopore made up of?

A

A tiny hole of 1nm immersed in conducting fluid

Across the membrane (synthetic or natural), there are electrons running across creating a charge

71
Q

Explain how a nanopore sequences DNA

A

Take long pieces of DNA

Use modified helicase at the entrance of the 1nm pore to make sure just one of the strands enters the pore

All the bases are aromatic, meaning they are insulators

Nucleotides passing through the hole creates a change in the current by reducing the number of electrons that can pass through the membrane

Each base is structurally different and will cause different changes in the current

72
Q

Can nanopore technology be used on RNA?

A

Yes

But is more of a quantitative approach at determining the concentration of proteins rather than the sequence

73
Q

What are the advantages of nanopores?

A

Portable

Abel to read kilobases at a time

Fast preparation time

CHeap

74
Q

What are the disadvantages of nanopores:?

A

High error rates

Slower than massively parallel sequencing

Can be affected by physical conditions like temperature due to the fragility of the lipid bilayer

75
Q

Applications of sequencing in cancer research

A

Whole genome sequencing

Transcriptome analysis

Methylome analysis

ChIP-seq

76
Q

What is the use of methylome analysis in cancer research?

A

Classification of tumour

Biomarker discovery

Genomics (9 decks)

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