Genome Sequencing Flashcards
What is the difference between a dNTP and ddNTP?
A ddNTP lacks a hydroxyl on the 3’ end
What is electrophoresis?
When the ddNTPs are added and bind to where it complements on the template strand then based on the different lengthed fragments the sequence can be determined
What do you add to a test tube for Sanger sequencing?
dNTP, ddNTP, taq polymerase, primers, and template DNA
What is the difference between the 96-well capillary and regular Sanger sequencing?
Sanger does not have as many wells as the capillary sequencing
What is an electropherogram?
The bar length tells you if the match is random or accurate
What if an electropherogram bar is short?
Could be random
What if an electropherogram bar is long?
The match is accurate
What is the shotgun approach?
Sequencing the genome in parts rather than as a whole
How is DNA fragmented?
Through random shearing and sonication
What is hierarchical shotgun sequencing?
The DNA is fragmented, then cloned using vectors, and then the cloned fragments are organized in a physical map to tell you the genome order
Why is the DNA cloned into vectors?
To ensure that there are multiple copies of the DNA fragment for cross comparison this can make sure that you are accurate in what you are doing
What is the promoter for?
Constituitive and inducible
What is the multicloning site?
A unique restriction site where the gene is inserted
What is the epitope tag?
Ensures that the protein is purified and localized to the right spot
What is the origin of replication?
Determines the copy number
What is a selectable marker?
Antibiotic resistance so to select for the cells you want you would add the antibiotic on the plate to kill the rest
What is a BAC?
Bacterial artificial chromosome
How can you use hybridization of probes to sequence a genome?
You take colonies from the plate using a robotic arm and a random sequence of a ssDNA probe is made and would hybridize to the overlapping fragment.
What is chromosome walking?
Where you generate a primer and the probe binds and you can determine the sequence and then you would repeat this process until you readch the end of the fragmentsa
What is the biggest disadvantage of chromosome walking?
It takes too long
How can you use restriction fingerprinting on BAC clones?
When a restriction enzyme recognizes its corresponding binding region it makes a double stranded break and leaves overhangs based on the overhangs of the DNA fragment you can organize the BAC clone fragments and order them
What is whole genome sequencing?
You fragment the entire genome then clone it and assemble the genome without generating a physical map
What are paired reads?
When an insert is sequenced from both ends using a universal primer
What is a universal primer?
A forward and reverse primer with the same sequence
What is coverage?
The number of times where you have to sequence a genome to have a high degree of accuracy
What is the genome coverage assumption?
That the sequencing reads are randomly distributed in the genome and the ability to sequence a region of the genome does not differ
How do you indentify overlaps?
They need to have >24 or more bp in common
What is a read?
The product of a sequencing reaction using DNA
What is a contig?
A continuous sequence made of overlapping reads with no gaps
What is a scaffold?
An ordered set of contigs usually derived from mate pairs
What is the problem with repeat regions in a genome?
It leads to misassembly because the system usually fails to recognize that it is in two regions
What is an intrinsic approach to finding unknown genes?
Looking for the coding sequences
What is the extrinsic approach to finding unknown genes?
Comparing to other genomes and looking for complementary
What is the difference between capillary and SGS?
SGS
1. Don’t need vector cloning
2. Read lengths shorter than Sanger
3. Need less genomic template
What is PCR?
A DNA amplification technique
1. Denature at 94 degees
2. Anneal at 54 degrees
3. Extend at 72 degrees
What principle does Roche 454 use?
Luciferase assay
Where is the DNA nicked in Roche?
At the beads 1 strand is stuck to the bead and binds to the linker while the other is not unless the other strand is bound to a linker
What happens when a dNTP matches the template in Roche 454?
There is light from the luciferase the amount of light corresponds to how many bind
What amplification technique is used in illumina sequencing?
The bridging reaction where the adaptor at one end joins to the flow cell where an existing adaptor is and generates replicates of the DNA attached to the adaptor
How does sequencing occur in SOLiD?
Using an 8-mer and the first 2 nucleotides have to bind and then another 8-mer is generated
Why is the primer in SOLiD offset by 1 nucleotide?
This makes it more accurate and allows for more coverage because you go over the same area more than once
What is the difference between SGS and TGS?
- No PCR
- Read lengths are longer
- The error rate is higher and the cost
In SMRT how is the incorporation of dNTPs measured?
By intensity of colour fluorescence
What happens when the dNTPs are passed through the Oxford nanopore?
It generates a change in current which is then measured
