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Bio 361 - Molecular Genome and Genomics > Midterm 1 - Notes 5 (Part 2) > Flashcards

Midterm 1 - Notes 5 (Part 2) Flashcards

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1
Q

What are the 5 steps in the genome sequencing principle?

A
  1. Start with a fragment that you need to cut into overlapping sequences
    - so you can sequence in one go
  2. Amplify each fragment so you have multiple copies
  3. Do individual sequences and get short reads that may have different lengths
  4. Get the sequence information one piece at a time
    - because we dont know where the original sequence came from
  5. Use overlapping segments to pin point where the original sequence came from
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2
Q

Why would you expect gaps in the genome sequencing?

A

Because they are randomly chosen

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3
Q

What is the sequence limited by?

A

Size

- few hundred bases

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4
Q

What is genome sequencing driven by?

A

Sequencing technology

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5
Q

What are 2 major challenges in genome sequencing?

A
  1. Throughput

2. Read length

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6
Q

What do you need in order to cover the whole genome?

A

A lot of read segments

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7
Q

Why do you need longer reads?

A

The longer they are the easier it is to assemble the sequence
- you get better quality

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8
Q

What are the 9 steps in the classical Sanger sequencing?

A
  1. Take a plasmid and separate it
  2. Generate a genomic generation line
  3. Cut up into DNA fragments
    - transform E. Coli to make a genomic library
  4. Clone into plasmid
    - multiple clones on plate
  5. Re-isolate multiplied plasmid to get it amplified in many copies
  6. Sequence one clone at a time
  7. Assemble sequence
  8. Close gaps
    - pre-finished sequence
  9. Assemble sequence
    - finished sequence
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9
Q

What are the 8 steps in NGS?

A
  1. Generate a genomic generation line
  2. Cut up into DNA fragments
  3. Immobilize on surface in parallel
  4. Amplify
  5. Sequence in parallel in one go
  6. Assemble sequence
  7. CLose gaps
    - pre-finished sequence
  8. Assemble sequence
    - finished sequence
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10
Q

What was the major difference between Sanger and NGS?

A

The fact that you can sequence in parallel in one go

- made things much faster

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11
Q

The original human genome project (4)

A
  1. Sanger sequencing
  2. Tool development
  3. 13 years (1990-2003)
  4. Over all cost = $2,700,000,000
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12
Q

What has decreased exponentially over the years?

A

The amount it is to sequence the human genome

- it is about $1,000 to do so

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13
Q

What are the 6 steps in Sanger sequencing sample preparation?

A
  1. Fragmentation of sample DNA
  2. Cloning into plasmids/ bacterial library
  3. Insert amplification
    - plasmid prep/ PCR
  4. Sequencing reactions
  5. Colony picker
  6. Pipetting robot
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14
Q

Colony picker

A

This machine takes pictures of the plate and can identify where the individual colonies were

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15
Q

Pipetting robot

A

Then can use this to go through sequences of pipetting to make things go faster

  • larger input
  • saving time and money
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16
Q

What are 4 advantages to Sanger sequencing?

A
  1. Targeting smaller genomic regions in a larger number of samples
  2. Validating results from NGS
  3. Identifying single disease-causing genetic variants
  4. Verifying plasmid sequences
17
Q

What are the 3 biggest challenges for NGS?

A
  1. Shorter reads
  2. Amount of date
    - assembly
  3. Quality of data
18
Q

What are 3 advantages of NGS?

A
  1. Massive parallel sequencing
  2. Very little sample prep
  3. High throughput
19
Q

What are the 5 steps in NGS?

A
  1. Immobilize DNA fragment
  2. Anneal primer
  3. Allow synthesis
  4. Detect which base was incorporated
  5. Go back to step 3 and repeat cycle
20
Q

What are 2 concepts/ major differences for NGS?

A
  1. Stop reaction after incorporating one base, measure results (use terminator)
  2. Monitor reaction on the go
    - instead of stopping after each base, let the polymerase do its work and measure as you go
21
Q

What are the 11 steps in Illumia sequencing?

A
  1. Random fragmentation of DNA into small pieces
  2. Ligate unique adaptors to each end
  3. Bind denatured fragments to flow cell surface
  4. Flow cell surface also contains oligos (primers) complementary to adapter in high density
  5. Perform bridge amplification
    - amplified segments attach to the surface
  6. Add primer complementary to one of the adaptors
    - similar to Sanger
  7. Add reversible terminators (each base labeled with a different fluorescent dye
    - fluorophone prevents incorporation of another base
    - -> prevents the bases from being elongated
  8. Add polymerase to add only one base, wash unincorporated nucleotides off
  9. Scan flow with fluorescent laser scaller, record first base
  10. Cleave off fluorescents dye, revents incorporated terminator to regular base
  11. Repeat steps 7-10 to capture subsequent bases
22
Q

What are 2 differences between Sanger and NGS?

A
  1. NGS is way shorter than Sanger

2. In NGS you can get multiple sequences at one time compared to Sanger where you only get one sequence at a time

23
Q

SMRT sequencing

A

Single Molecule Real Time sequencing

24
Q

What is the idea behind SMRT sequencing?

A

To immobilize the DNA polymerase, not the DNA

25
Q

What do they do in SMRT sequencing instead of immobilizing and adding a probe?

A

They stick the DNA polymerase on the surface and let it float and the polymerase will catch one piece of DNA and continue to generate a complementary strand

26
Q

What do they think will happen in theory for SMRT sequencing?

A

It will produce very long strands

27
Q

What are 2 problems with SMRT sequencing?

A
  1. You cannot stop the polymerization after one incorporation, because the DNA polymerase will dissociate from the strand and a new polymerase will would add on and then you would get repeated sequences
  2. You have to distinguish between a base that is incorporated from one that is diffusing by
    - incorporation takes longer but still just a few milliseconds and you will have way more diffusing than incorporating bases
28
Q

What does PacBio: SMRT not do?

A

Does not incorporate amplification

29
Q

Where is DNA polymerase immobilized in PacBio: SMRT?

A

At the bottom of the wells

30
Q

What are 3 benefits to SMRT sequencing capture?

A
  1. Detection is continuous
  2. Very fast
  3. Can go on very long
31
Q

What is a problem with SMRT sequencing capture?

A

It comes with a very high error rate

32
Q

Where does the sequence get labelled in SMRT sequencing?

A

At the end of the triphosphate

33
Q

What is the sample preparation for SMRT? (3)

A
  1. Fragment DNA
  2. Ligate hairpin adapter to each end
  3. Denature DNA
    - generates circular single stranded DNA
    - the same DNA will be sequenced multiple times (this is so it can reduce its errors)
34
Q

Hairpin adaptor (2)

A
  1. Continuous DNA that can bind to both ends of the DNA

2. If you denature it you get a circular piece of DNA

Bio 361 - Molecular Genome and Genomics (35 decks)

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