Lecture 12

Question 1

what are the three most used third generation sequencing platforms?

Answer 1

1. HeliScope
2. Pacific Biosciences
3. Nanopore

Question 2

what is third generation sequencing not widely used in?

Answer 2

diagnostics → accuracy of the approach is limited

Question 3

what are the principle features of third generation sequencing?

Answer 3

1. platforms are characterized by very high sensitivity and it is possible to sequence and to read the sequences of one single molecule of RNA or DNA
2. characterized by high cost
3. we can have an easier preparation of the sample because it is not necessary to have amplification or library preparation
4. longer length of reads generated
5. faster fun for analysis
6. easier analysis in the primary step (often preformed directly on the platform and at the end of the analysis we already have the list after the comparison of the reference genome)

Question 4

what was the first third generation platform to be commercialized?

Answer 4

HeliScope

Question 5

what is HeliScope based on?

Answer 5

the sequencing by synthesis approach, but in this case without the amplification and the creation of the library

Question 6

STUDY THE PROCESS OF HELISCOPE SEQ

Question 7

what is a simplified version of how HeliScope works?

Answer 7

each cycle consists of the incorporation mediated by DNA polymerase of a single species of nucleotides labelled with Cy5 (reversible terminators), in the detection of the fluorescence emitted from the entire array and in the chemical removal of the fluorphore

Question 8

what does the HeliScope record?

Answer 8

when on each cluster fluorescence is released, and in that case the platform knows which kind of nucleotide is added in that specific moment

Question 9

what are the three main features of HeliScope?

Answer 9

1. low amount of DNA / RNA
2. we don’t have an error in the C-G rich regions or also in the polymeric regions
3. good for the use of paraffin (usually difficult to do because the DNA or nucleic acids are damage and is quit difficult to perform sequencing) - success rate is good and we obtain billions of reads per sequence

Question 9

what are the three main features of HeliScope?

Answer 9

1. low amount of DNA / RNA
2. we don’t have an error in the C-G rich regions or also in the polymeric regions
3. good for the use of paraffin (usually difficult to do because the DNA or nucleic acids are damage and is quit difficult to perform sequencing) - success rate is good and we obtain billions of reads per sequence

Question 10

what are the two main issues with HeliScope?

Answer 10

produces really short reads (55 bp) and has a high error rate

Question 10

what are the two main issues with HeliScope?

Answer 10

produces really short reads (55 bp) and has a high error rate

Question 11

what are the main features of Pacific Biosciences sequencing?

Answer 11

1. zero-mode waveguide (ZMW)
2. modified DNA polymerase for the modified dNTPs incorporated
3. dNTPs are labelled with fluorescence but they are not terminator dNTPs, so the synthesis goes on
4. can produce reads with a mean length from 4,200-8,500 (up to 30,000)
5. high accuracy (99.99%) with a low error rate

Question 12

how does PacBio work?

Answer 12

polymerase is fixed on the solid support and we have the flow of DNA molecules in the polymerase and the addition of the nucleotide while the specific bases are in the polymerase

Question 13

what does PacBio use?

Answer 13

fluorescent phospholinked nucleotides

Question 14

what are the four steps of PacBio?

Answer 14

1. a modified nucleotide is incorporated define the synthesis by the DNA polymerase
2. the fluorescent signal is released and each of the different nucleotides is labeled with a different fluorophore
3. the fluorophore goes away with a time of dark signal
4. the DNA template shifts to the following nucleotide
5. the cycle starts again, and another modified nucleotide is incorporated

Question 15

what is nano pore sequencing based off of?

Answer 15

the use of a very thin modified membrane → a DNA molecule has a negative charge, so if we have a membrane with a different potential among the two faces of the membrane and there is a DNA molecule on one side of the membrane, there is a change in the potential on the other side of the positive pole, the DNA can pass through the membrane

Question 16

how is the membrane used in nanopore sequencing modified?

Answer 16

modified so it contains a lot of nanopores with a diameter 1.5-2 nm long where the DNA can pass through when a specific nucleotide is in the center of that pore, and we have a change in voltage that is recorded on that membrane → different voltage change based on the kind of nucleotides that is present

Question 17

what type of flow is required for nanopore sequencing?

Answer 17

unidirectional → other we read the same base two time

Question 18

what is the plus of nanopore sequencing?

Answer 18

it can sequence low DNA fragments with very good accuracy

Question 19

what is the MinION able to do?

Answer 19

can be directly connected to a PC, and they are connected to an internal cloud to store all of the raw data → can perform sequencing ANYWHERE as long as it is connected to the internet

Question 20

where were the MinION nanpore sequencer the first to be used?

Answer 20

inside the international space station

Question 21

what are the two most famous databases with the genetic sequences of different populations?

Answer 21

1000 genome project and the gnomAD database

Question 22

what is one positive related to the ease of sequencing the human genome?

Answer 22

someone was able to buy a commercial kit and performed his own DNA analysis, and discovered his predisposition for cardiovascular disease - because of this knowledge he was able to take precautions against it

Question 23

what is a major downside to the ease of sequencing the human genome?

Answer 23

incidental findings → genetic information is gained and people start to make decisions without the use of genetic counseling which can lead to dangerous and detrimental decisions

Question 24

outside of the medical field, what else has genome sequencing such as nanopore been used for?

Answer 24

to solve criminal investigations - child went missing and was re-located years later through 23andme results that allowed her to find her real parents