Midterm 1 - Notes 5 (Part 2) Flashcards
What are the 5 steps in the genome sequencing principle?
- Start with a fragment that you need to cut into overlapping sequences
- so you can sequence in one go - Amplify each fragment so you have multiple copies
- Do individual sequences and get short reads that may have different lengths
- Get the sequence information one piece at a time
- because we dont know where the original sequence came from - Use overlapping segments to pin point where the original sequence came from
Why would you expect gaps in the genome sequencing?
Because they are randomly chosen
What is the sequence limited by?
Size
- few hundred bases
What is genome sequencing driven by?
Sequencing technology
What are 2 major challenges in genome sequencing?
- Throughput
2. Read length
What do you need in order to cover the whole genome?
A lot of read segments
Why do you need longer reads?
The longer they are the easier it is to assemble the sequence
- you get better quality
What are the 9 steps in the classical Sanger sequencing?
- Take a plasmid and separate it
- Generate a genomic generation line
- Cut up into DNA fragments
- transform E. Coli to make a genomic library - Clone into plasmid
- multiple clones on plate - Re-isolate multiplied plasmid to get it amplified in many copies
- Sequence one clone at a time
- Assemble sequence
- Close gaps
- pre-finished sequence - Assemble sequence
- finished sequence
What are the 8 steps in NGS?
- Generate a genomic generation line
- Cut up into DNA fragments
- Immobilize on surface in parallel
- Amplify
- Sequence in parallel in one go
- Assemble sequence
- CLose gaps
- pre-finished sequence - Assemble sequence
- finished sequence
What was the major difference between Sanger and NGS?
The fact that you can sequence in parallel in one go
- made things much faster
The original human genome project (4)
- Sanger sequencing
- Tool development
- 13 years (1990-2003)
- Over all cost = $2,700,000,000
What has decreased exponentially over the years?
The amount it is to sequence the human genome
- it is about $1,000 to do so
What are the 6 steps in Sanger sequencing sample preparation?
- Fragmentation of sample DNA
- Cloning into plasmids/ bacterial library
- Insert amplification
- plasmid prep/ PCR - Sequencing reactions
- Colony picker
- Pipetting robot
Colony picker
This machine takes pictures of the plate and can identify where the individual colonies were
Pipetting robot
Then can use this to go through sequences of pipetting to make things go faster
- larger input
- saving time and money
What are 4 advantages to Sanger sequencing?
- Targeting smaller genomic regions in a larger number of samples
- Validating results from NGS
- Identifying single disease-causing genetic variants
- Verifying plasmid sequences
What are the 3 biggest challenges for NGS?
- Shorter reads
- Amount of date
- assembly - Quality of data
What are 3 advantages of NGS?
- Massive parallel sequencing
- Very little sample prep
- High throughput
What are the 5 steps in NGS?
- Immobilize DNA fragment
- Anneal primer
- Allow synthesis
- Detect which base was incorporated
- Go back to step 3 and repeat cycle
What are 2 concepts/ major differences for NGS?
- Stop reaction after incorporating one base, measure results (use terminator)
- Monitor reaction on the go
- instead of stopping after each base, let the polymerase do its work and measure as you go
What are the 11 steps in Illumia sequencing?
- Random fragmentation of DNA into small pieces
- Ligate unique adaptors to each end
- Bind denatured fragments to flow cell surface
- Flow cell surface also contains oligos (primers) complementary to adapter in high density
- Perform bridge amplification
- amplified segments attach to the surface - Add primer complementary to one of the adaptors
- similar to Sanger - Add reversible terminators (each base labeled with a different fluorescent dye
- fluorophone prevents incorporation of another base
- -> prevents the bases from being elongated - Add polymerase to add only one base, wash unincorporated nucleotides off
- Scan flow with fluorescent laser scaller, record first base
- Cleave off fluorescents dye, revents incorporated terminator to regular base
- Repeat steps 7-10 to capture subsequent bases
What are 2 differences between Sanger and NGS?
- 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
SMRT sequencing
Single Molecule Real Time sequencing
What is the idea behind SMRT sequencing?
To immobilize the DNA polymerase, not the DNA
What do they do in SMRT sequencing instead of immobilizing and adding a probe?
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
What do they think will happen in theory for SMRT sequencing?
It will produce very long strands
What are 2 problems with SMRT sequencing?
- 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
- 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
What does PacBio: SMRT not do?
Does not incorporate amplification
Where is DNA polymerase immobilized in PacBio: SMRT?
At the bottom of the wells
What are 3 benefits to SMRT sequencing capture?
- Detection is continuous
- Very fast
- Can go on very long
What is a problem with SMRT sequencing capture?
It comes with a very high error rate
Where does the sequence get labelled in SMRT sequencing?
At the end of the triphosphate
What is the sample preparation for SMRT? (3)
- Fragment DNA
- Ligate hairpin adapter to each end
- Denature DNA
- generates circular single stranded DNA
- the same DNA will be sequenced multiple times (this is so it can reduce its errors)
Hairpin adaptor (2)
- Continuous DNA that can bind to both ends of the DNA
2. If you denature it you get a circular piece of DNA
