MBB 267 Week 1: RMC 2 Flashcards
What was the problem with sanger sequencing?
Does Not scale very well = each capillary produces one read at a time
Advantages and disadvantages of sanger sequencing
Advantages: long (~800bp) and high quality reads Disadvantages: One molecule is sequenced at a time so very expensive
Features of Illumina sequencing
One run produces ~8 billion sequence reads and relatively cheap ($1000). However, maximum read length is 125bp.
Sanger vs. Illumina sequencing
Differences:
- Illumina produces shorter reads but generates many reads simultaneously
- Both use sequencing by synthesis
- Illumina uses fluorescent bases to detect each base
What is the process of illumina sequencing?
The key stages of the Illumina sequencing process are:
- Genomic DNA extraction
- DNA fragmentation:fragment into ~500bp using sonication or enzymatically.
- Library preparation (addition of adapter molecules): Adapters are added to both ends of all fragments. Adapters allow us to amplify and sequence the DNA fragments
- Cluster generation (bridge amplification)
- Sequencing by synthesis
- Data analysis
How does bridge amplification work?
Start off with a flow surface cell, which is covered with 2 different types of primer sequences which are complementary to the adapters. The template construct is melted, and the single-stranded molecules are washed over the surface of the flow cell. The adapters at the ends bind to the single stranded molecules and then DNA polymerase is added which extends the flow cells strands. The original template is washed away. The newly synthesised strand has at its end another adapter sequence, which is complementary to the second primer on the flow cell surface. The molecule can form a bridge-like structure allowing the adapter which is not attached to hybridise to a neighbouring primer. Again a polymerase allows the primer to be extended, generating a second strand. When the replication process is complete, the temperature is increased, giving two copies of the original template, each attached to the flow cell surface. This bridge amplification process repeats many times to form a cluster of sequences.
How does illumina use sequencing by synthesis?
Illuminas sequencing by synthesis approach exploits reversible terminator nucleotides. These include a detachable fluorescent label which work like ddNTPs as they block to prevent chain extension, but the block can be chemically removed: An endonuclease is used to cleave off one of the strands meaning that all remaining strands in the cluster have the same sequence, The reversible terminator nucleotides are added and just one will hybridise to its template. A laser is used to excite the fluorophore and the camera will detect the colour and hence the base that is incorporated. The chain block is removed for the ndt sequencing cycle.
What is illuminas paired-end sequencing?
Since the DNA fragments are bound to 2 adapters, these adapters have 1 primer sequence each. Meaning we can read the same strand twice because the second primer is on the other adapter. A third primer is sometimes used to sequence the index sequences which acts as a barcode for each adapter. This allows different samples to be distinguished from others.
How does illumina paired-end sequencing work?
First we melt our construct to get rid of the previously synthesised strand. A round of bridge PCR is then performed to generate the complementary strand. Original/ old strand can be removed via an endonuclease. The second read occurs.
What does next generation sequencing require?
Requires amplification of the template DNA fragments.
