Lesson 2

Question 1

What are the 3 steps of the basic sequencing workflow?

Answer 1

Sample preparation, detection, and analysis

Question 2

What are the 4 steps of library template preparation?

Answer 2

DNA fragmentation, end repair, da tailing and adapter ligation

Question 3

What are the 2 reasons why DNA fragmentation is absolutely necessary?

Answer 3

It is impossible to efficiently amplify molecules longer than 1000 bp and NGS sequencers are not able to sequence long reads

Question 4

What is the most used technique for DNA fragmentation?

Answer 4

Sonication

Question 5

What is characterized by the addition of a phosphate at the 5’ end of the molecule or filling by DNA polymerase?

Answer 5

End repair

Question 6

What is added in da tailing?

Answer 6

A nontemplate A at the 3’ end of each strand

Question 7

What is a sequence which allows for the hybridization of DNA fragments to the flow cell of the sequencer?

Answer 7

Adapter

Question 8

What is attached to the flowcell chip?

Answer 8

DNA oligomers complimentary to the acceptor sequence

Question 9

Describe a single index adapter

Answer 9

DNA is inserted in the center surrounded by 2 adapter sequences called p5 and p7 and a 6 letter “barcode” called the I7 index

Question 10

Describe a dual index

Answer 10

The same as a single index but with 2 indexs added, one at the 5’ end and the other at the 3’ end when the fragment needs to be sequenced from both sides

Question 11

Describe a dual index UMI

Answer 11

Same as a duel index but with a UMI (a particular barcode not detected in the sample identifying with a molecule or a cell

Question 12

What type of pcr does ion torrent sequencing use?

Answer 12

Emulsion pcr

Question 13

What type of pcr does illumina sequencing use?

Answer 13

Polony pcr

Question 14

When performing emulsion pcr what do the oil droplets created contain?

Answer 14

The fragment, primers necessary to perform the pcr, enzymes + cofactors, and a bead coated by oligos complimentary to the the adapter sequence

Question 15

What is sequencing depth?

Answer 15

The number of reads obtained

Question 16

What is the Max read length for ion torrent sequencing?

Answer 16

200 bp

Question 17

What is the throughput in ion torrent sequencing?

Answer 17

The number of bases you are going to acquire

Question 18

What is ion torrent sequencing based on the addition of?

Answer 18

A new nucleotide

Question 19

When is the new nucleotide added in ion torrent sequencing?

Answer 19

During the synthesis of the complimentary DNA strand

Question 20

What specifically are the wells of an ion torrent chip detecting?

Answer 20

Defects a change in pH due to the release of H+ ions occurring after the incorporation of the nucleotide inside the DNA

Question 21

What does polomy pcr stand for?

Answer 21

Polymerase colony

Question 22

What type of sequencing is colony pcr?

Answer 22

Sequencing by synthesis

Question 23

Describe sequencing by synthesis (polony pcr)

Answer 23

The DNA polymerase amplifies the molecule and if nucleotides are coupled with fluorphores, it is possible to read the sequence

Question 24

What are two scenarios where colony pcr is useful?

Answer 24

When there are places in the genome with long dimer repeats and to determine now many base pairs are between the 1st and 2nd reads

Question 25

What is the main difference between 2nd and 3rd generation sequencing?

Answer 25

3rd generation sequencing is able to achieve a much higher read length compared to NGS

Question 26

What are the two main 3rd gen sequence companies?

Answer 26

Pacific biosciences and Oxford nano por technology

Question 27

What is unique about pacific biosciences?

Answer 27

Augmentation is still required but no amplification is needed

Question 28

List the 4 steps of oxford nanopore sequencing:

Answer 28

1) one protein unzips the DNA helix into 2 strands
2) a second protein creates a pore in the membrane and holds an “adapter” molecule
3) a flow of ions through the pore creates a current → each base blocks the flow to a different degree, altering the current
4) the adaptor molecule keeps bases in place long enough for them to be identified electronically

Question 29

What are the main advantages of nanopore sequencing?

Answer 29

There is no need to amplify the molecules and since no PCR required, it could lead to the achievement of a huge read length

Question 30

What is the main issue in nanopore sequencing

Answer 30

During BAs coding there is a high % error (10%) due to the use of ai