MBB 267 Week 2: RMC 3 Flashcards
Advantages of second generation sequencing?
Advantages over Sanger sequencing:
- Massively parallel, a single run generates millions of -sequences
- Much cheaper per base
- “Built-in” shotgun sequencing without a cloning step
Disadvantages of second generation sequencing?
Disadvantages:
- Library preparation is quite expensive and slow
- Amplification of DNA fragments is required, which can introduce biases (e.g. GC-rich sequences often do not amplify efficiently using PCR)
- Read lengths are quite short (maximum of 300bp)
Examples of third generation sequencing?
PacBio and Oxford Nanopore
Advantages of third generation sequencing?
Advantages over Illumina sequencing:
- Single molecule sequencing - no amplification required
- Real time sequencing - data is generated during the run
- Ultra-long read lengths up to 50kb (PacBio) or 2Mb (nanopore)
- Can directly identify base modifications such as methylation
disadvantages of third generation sequencing?
Disadvantages:
-Fewer reads per run than Illumina
-More expensive per base
I-ndividual reads have a high error rate (although consensus accuracy is good)(because ur using 1 molecule while second generation sequencing use many molecules)
What are Zero-mode Waveguides (ZMWs)?
A PacBio SMRT cell consists of an aluminium surface covered with around 150,000 wells called Zero mode waveguides.
How does Zero-mode Waveguides (ZMWs) work?
During library prep, the double-stranded template DNA is bound to a DNA polymerase and a sequencing primer. The polymerase is then immobilized at the bottom of a well. One polymerase is present per well. A laser is used to excite the fluorescently-labelled nucleotides. The ZMW only allows the wavelength to penetrate a small distance into the well, meaning that a very small volume is illuminated (zeptolitres). The ZMW acts as “the world’s most powerful microscope” allowing the signal from a single fluorescently-labelled nucleotide to be detected.
How does PacBio sequencing work?
Fluorescently labelled nucleotides are used to give a signal. The label is attached in a way that when the DNA polymerase attaches the nucleotides to the other nucleotide, the label is cleaved. This prolonged period gives a detectable signal. This gives a pulse reading.
What are SMRT-bell adapters?
PacBio library prep results in a double stranded template DNA molecule, with “SMRT-bell” adapters at either end. These are hairpin loops which connect the two strands of the template DNA, resulting in a single continuous loop of DNA. A primer which is complementary to one of the adapters is annealed. During sequencing, the polymerase extends from the primer around the DNA loop, displacing the complementary strand as it goes. The polymerase continues to displace the complementary strand when it reaches the original primer, so sequencing can continue around the loop. This keeps repeating until the primer falls off.
How does consensus sequencing work?
The polymerase reads (from the SMRT-bell ) are used to produce a consensus sequence as there will be many subreads of the same sequence.
What are the key features of PacBio sequencing?
Features:
- Like Illumina, PacBio is an example of sequencing by synthesis
- PacBio also uses fluorescently-labelled nucleotides, however the label is on the phosphate group, not the base, so it is removed when the strand is extended
- Unlike Illumina, PacBio sequences a single molecule at a time, so no PCR amplification is required
- No pause is required between base incorporations, meaning that data can be accumulated continuously. This is referred to as Single Molecule Real Time sequencing (SMRT sequencing)
- PacBio can detect fluorescence from a single molecule due to the use of Zero-Mode Waveguides (ZMWs)
- Individual read error rates are fairly high (13-15%), but this can be compensated using circular consensus sequencing (CCC)
What is DNA methylation?
DNA methylation involves the addition of a methyl group to the C5 carbon residue of cytosine. This is distinct from histone methylation. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription
Uses of DNA methylation?
Methylation is an important epigenetic modification in higher eukaryotes, involved in many processes:
- Regulation of gene expression
- X chromosome inactivation
- Silencing of germline-specific genes and repeat regions
- Repeat regions
- Imprinting (use of methylation to distinguish maternal and paternal alleles). This is important in the genetic conditions Angelman’s syndrome (associated with a deletion of a region of the maternal copy of chromosome 15) and Prader-Willi syndrome (associated with a deletion of the same region of the paternal copy)
- In bacteria, methylation is used to distinguish “self” DNA from “non-self”, e.g. from phage. The non-self DNA can be digested using restriction endonucleases, acting as an immune system. Methylation also plays an important role in controlling bacterial DNA replication, limiting it to a single replication per cell cycle
How does direct methylation sequencing in PacBio work?
PacBio single molecule real-time (SMRT)-sequencing allows methylated bases to be distinguished from unmethylated ones. This includes methylated adenine as well as cytosine. The presence of a methylated base delays the progress of the polymerase, and can be detected by analysis of the polymerase kinetics.
How does Oxford Nanopore work?
Oxford Nanopore sequencing uses pore proteins embedded within an artificial membrane which is electrically insulating. The pore protein is currently CsgG from E.coli. The motor protein pushes a single strand of the DNA through the pore, and this results in a change in the electrical current flowing through the pore. Sequencing does not happen a single base at a time - several bases can pass through the pore at the same time and the short sequences of e.g. 5 bases each have their own characteristic signal. There are software packages which convert this “squiggle” into DNA sequences.
