VL 1b: NGS

Question 1

ILLUMINA

Answer 1

• Ligation of adapters to each end of the ssDNA molecule.
• Single strands are coupled to glass slides, via adaptors.
• PCR done on slide to form “PCR colonies” or “polonies”.
  -> bridge amplifictaion (bridg forms naturally due to movement & complementarity
• During sequencing, nucleotides are blocked, so no more than one can be incorporated per cycle.
• Four fluorescent dyes, detection of pictures.

-> keeping primary data is not possible (too many pictures)

Amount of data increased, but computers did not (slowly alining again today)

Question 2

Nanopore

Answer 2

• Proposed and started in the early 1990’s in Santa Cruz and Harvard.
• Based on threading a single strand of DNA through a microscopic hole in a membrane.
• Creating an electric field across the membrane causes the DNA to pass through. (The negatively charged DNA is attracted to the positively charged electrode, causing it to pass through the nanopore)
• Measuring the electrical properties of the hole (capacitance), should tell you which base is passing through it.
• Resolution has initially proved to be a bit of a problem…but it is really good by now.
• measures each position twice, first one single strand and then the other
• cheap & portable
• Long range sequencing/ Extremely long reads (up to 150 kb, even 1 MB have been reported).
• Directly accessing original DNA molecules.
• Capable of distinguishing modified bases directly.

Question 3

PacBio

Answer 3

• SMRT- cell with ZMW (Polymerase at the bottom)
• Nucleotides labeled with flourescent dye
• light change -> base calling
• Strictly single molecule reaction monitoring. (SMRT sequencing monitors the incorporation of nucleotides by a single DNA polymerase molecule in real-time, as it synthesizes a complementary strand of DNA.)
• Diffusion, therefore no washing: cheap on reagents.
• No stop-and-go synthesis as in other systems.
• Recent upgrade to 8x more reads per run.
• Read length is up to ~40,000 bases.
• Initially high error rate, now reduced, thanks to circular sequencing (PacBio HiFi – high fidelity).
• cicular molecules to read each position several times -> lower error rate now
• Highly competitive for long reads.

Question 4

Selective sweep

Answer 4

A selective sweep is a process where a beneficial mutation rapidly increases in frequency within a population, reducing genetic variation in the surrounding region of the genome.

z.B. Birkenspanner (peppered moth)

-> very hard selection fixated mutation very fast