20.02.18 Third generation Sequencing

Question 1

What is third generation sequencing (TGS)

Answer 1

-Single molecule sequencing. Aims to sequence single DNA molecules without the need for PCR-based amplification.

Question 2

Possible uses of TGS

Answer 2

Sequencing long repetitive elements and larger structural variations, which short read technologies struggle with.

Question 3

Advantages of TGS

Answer 3

• 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)

Question 4

Three types of TGS technology

Answer 4

1. Seqeuncing by synthesis
2. Nanopore
3. Synthetic long read

Question 5

Review of sequencing by synthesis TGS technology

Answer 5

• 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

Question 6

Review of nanopore TGS technology

Answer 6

• 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.

Question 7

Review of synthetic long read TGS technology

Answer 7

• 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.

Question 8

How does single molecule real time (SMRT) sequencing work

Answer 8

• 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.

1. Template fragments are process and ligated to hairpin adapters at each end, resulting in a circular DNA molecule.
2. A single DNA polymerase is anchored to the bottom surface of each ZMW
3. Circular DNA enters ZMW and is processed by polymerase
4. Fluorescently labelled nucleotides are flooded
5. When polymerase incorporates a nucleotide. DNA pol cleaves the bond attached to the fluorophore. Dye moves away and signal returns to baseline.
6. Cycle repeated

Question 9

Advantages of SMRT

Answer 9

• Fast
• Circular template allows polymerase to transverse the length many times, to give high coverage. Needed as error rate is ~15% per read through.

Question 10

Disadvantages of SMRT

Answer 10

• Limited throughput, due to limited number of ZMW.
• Longer molecules take longer to pass through ZMW
• Expensive

Question 11

Application of SMRT

Answer 11

Rapid identification of infectious disease agents.

Question 12

What is FRET

Answer 12

• 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.

Question 13

Benefits of FRET

Answer 13

Polymerase is not bound to a solid substrate so can be exchanged mid run, replacing damaged polymerases extending net read-length capability.

Question 14

What is nanopore technology

Answer 14

• 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.

Question 15

What are biological nanopores

Answer 15

Transmembrane protein channels inserted into a substrate

Question 16

Examples of biological nanopores

Answer 16

• Alpha-hemolysin: exotoxin secreted from staphylococcus aureus. Stable, but only can be used for ssDNA or RNA, due to the small pore size. Reads 3-4 nucleotides simultaneously. Higher error rate.
• MspA: porin A channel from a mycobacterium. Stable, shorted blockade region so better resolution. Reads 3-4 nucleotides simultaneously. Higher error rate.
• Bacteriophage phi29. Largest pore so can measure dsDNA, DNA complexes and proteins.

Question 17

What is MinION

Answer 17

• Nanopore sequencer from Oxford nanopore technologies.
• DNA is linked to an enzyme to unwind dsDNA so a single strand is fed into the nanopore
• Flow of ions through pore creates a current. Each base blocks the flow to different degrees, altering the current. Identified electronically.
• Read as K-mers (rather than individual nucleotides). Therefore 1000 possible signals, leads to a higher error rate.

Question 18

What are synthetic nanopores

Answer 18

• Graphene nanopore.
• Chemically inert, single atom thick honeycomb lattice of carbon with high electrical conductivity.
• Can be very thin to allow measuring variations between single DNA molecule

Question 19

Benefits of synthetic nanopores over biological

Answer 19

• More stable than biological nanopores.
• More control over pore size and lower sensitivity to external parameters such as pH, temp, salt concentration.
• Better suited to upscaling and integration onto chips.

Question 20

Disadvantages of synthetic nanopores compared to biological

Answer 20

-More noise

Question 21

What is synthetic long read

Answer 21

• DNA is fragmented into large segments (~10kb) and partitioned into microtitre wells or an emulsion
• Fragments are then sheared and barcoded and sequenced on existing instruments.
• Fragments with the same barcode originate from the same large fragment so sequence can be reassembled.

Question 22

Examples of Synthetic long read technology

Answer 22

• Illumina synthetic long read: kb molecules of DNA are clonally amplified and barcoded before short read sequencing. Accurate but relies on long-range amplification and biased to regions of high GC content or tandem repeats.
• 10X Genomics: Oil emulsion and multiple displacement amplification (MDA) to amplify and ligate barcodes onto large DNA molecules (100kb). Fragments are partitioned into micelles each with a specific barcode. Gaps in coverage but overcome by ensuring there are many long fragments from the same genomic region in the initial preparation. Produces de novo assemblies that can phase megabase regions of genome, for structural variation/ haplotype analysis.

Question 23

What is third generation mapping

Answer 23

-Determine large-scale sequence structure of DNA without sequencing every base.

Question 24

Examples of third generation mapping

Answer 24

-Irys system from BioNano Genomics: Iris is an optical mapping system, using fluorescently tagged probes attached at nicked restriction digest sites to fingerprint long DNA molecules. After imaging, the per-molecules fingerprints are assembled into larger optical maps, spanning megabases of chromosome. Can be biased, due to incomplete nicking of DNA that causes a proportion of digest sites to remain unlabelled and fragile sites where multiple nicks in close proximity cause DNA shearing.

Question 25

Challenges for the future

Answer 25

- Time: most current systems do not generate enough data fast enough for a rapid response (e.g. in a hospital setting for monitoring infection) - Storage and bioinformatic solutions are required to handle volume of data generated - Utility and ethics: direct to consumer testing and whether consumer understands personal impact of results and false positives/negatives