20.02.18 Third generation Sequencing Flashcards
What is third generation sequencing (TGS)
-Single molecule sequencing. Aims to sequence single DNA molecules without the need for PCR-based amplification.
Possible uses of TGS
Sequencing long repetitive elements and larger structural variations, which short read technologies struggle with.
Advantages of TGS
- Small amount of starting material needed
- Higher throughput
- Lower cost per base, so higher coverage possible.
- Longer read lengths allow enhanced de novo assembly, chromosome phasing, CNV detection, identification of chimeric/alternatively spliced transcripts.
- Sequencing of repetitive elements (more contiguous reconstructions of the genome)
- More uniform coverage of genome, less sensitive to GC content
- Potential to detect epigenetic modifications (methlation)
Three types of TGS technology
- Seqeuncing by synthesis
- Nanopore
- Synthetic long read
Review of sequencing by synthesis TGS technology
- In SGS (second generation sequencing) Sequence information is generated by polymerase that copies a DNA strand.
- In TGS, technology directly “reads” original DNA/RNA molecule. Overcomes biases introduced by PCR amplification and dephasing
- E.g. Single molecule real time (SMRT) sequencing - Pacific Bioscience
Review of nanopore TGS technology
- Single DNA molecule is threaded through a nanopore (biologic or synthetic) and individual bases are detected as they pass through the nanopore.
- E.g. Oxford Nanopore.
Review of synthetic long read TGS technology
- Partition large DNA fragments into microtitre wells or an emulsion with few fragments per partition.
- In each partition fragments are sheared and barcoded.
- Uses existing short-read sequencing, after which the reads with the same barcode are assembled as they must be derived from the same original large fragment.
- E.g. 10X Genomics.
How does single molecule real time (SMRT) sequencing work
- Monitors polymerase activity whilst incorporating differently labelled nucleotides into DNA strand.
- Uses zero-mode waveguide (ZMW): a hole measuring 30-70nm in diameter.
- Small size of hole prevents visible laser light from passing entirely through.
- Template fragments are process and ligated to hairpin adapters at each end, resulting in a circular DNA molecule.
- A single DNA polymerase is anchored to the bottom surface of each ZMW
- Circular DNA enters ZMW and is processed by polymerase
- Fluorescently labelled nucleotides are flooded
- When polymerase incorporates a nucleotide. DNA pol cleaves the bond attached to the fluorophore. Dye moves away and signal returns to baseline.
- Cycle repeated
Advantages of SMRT
- Fast
- Circular template allows polymerase to transverse the length many times, to give high coverage. Needed as error rate is ~15% per read through.
Disadvantages of SMRT
- Limited throughput, due to limited number of ZMW.
- Longer molecules take longer to pass through ZMW
- Expensive
Application of SMRT
Rapid identification of infectious disease agents.
What is FRET
- Fluorescence resonance energy transfer
- DNA polymerase is tagged with a fluorophore that when brought into close proximity to a nucleotide (tagged with an acceptor fluorophore), emits a FRET signal
- Fluorophore is then removed ready for addition of next nucleotide.
Benefits of FRET
Polymerase is not bound to a solid substrate so can be exchanged mid run, replacing damaged polymerases extending net read-length capability.
What is nanopore technology
- Where ssDNA molecules are electrophoretically driven through a nanoscale pore.
- In a salt buffer solution where an electrical potential is applied creating an electrical current though the pore.
- As molecules move through the pore they can change the ionic current. The physical and chemical properties can lead to current blockades.
- Due to the length of the pore the shift in voltage is caused by a string of bases (not just one nucleotide)
- Nanopores can be synthetic or biological.
What are biological nanopores
Transmembrane protein channels inserted into a substrate
