Lecture 3

Question 1

Define “massive parallel sequencing” in NGS:

Answer 1

Massive: several regions at once; parallel: several samples at a time

Question 2

What are the 2 main NGS platforms currently used?

Answer 2

Ion torrent and illumina

Question 3

What is ion torrent also referred to as?

Answer 3

“Label-free sequencing” - fluorescence or spikes of light are not used

Question 4

What does ion torrent measure?

Answer 4

Changes in ph

Question 5

What is ion torrent characterized by?

Answer 5

High accuracy and good coverage

Question 6

How long are the reads sequenced by ion torrent?

Answer 6

About 200 bp

Question 7

How long are the average Sanger sequence reads?

Answer 7

600-800 bp up to 1000

Question 8

What is ilumina characterized by?

Answer 8

Bridge amplification

Question 9

How is illumina visualized?

Answer 9

By the use of fluorescence → each nucleotide is linked to a different fluorophore which emits a unique signal

Question 10

Describe the library structure of an illumina sample:

Answer 10

A DNA insert with “read1” and “read2” ( primers similar to the forward and reverse primers in Sanger) on either side, two indexes on either side that serve as an 8 bp “barcode” exclusive to each sample, and 2 adaptors complimentary to those linked on the flow cell called p5 and p7

Question 11

What is the process called in which all samples collected by illumina sequencing are pulled together to observe associations between the obtained sequence and the sorted samples?

Answer 11

Demultiplexing → each read is associated to a unique sample

Question 12

What is cluster generation?

Answer 12

Amplification of the flow cell

Question 13

What is a quality score?

Answer 13

A prediction of the probability of an error in base calling

Question 14

When measuring Phred quality score, what probability corresponds to high accuracy?

Answer 14

Low probability

Question 15

What is read depth?

Answer 15

The total number of bases sequenced and aligned at a given reference base position

Question 16

What is coverage defined as?

Answer 16

The average number of sequenced bases that align to each base of the reference DNA → ex: a whole genome sequenced at 30x coverage means that each base in that genome analysis was sequenced 30 times on average

Question 17

What does NGS accuracy depend on?

Answer 17

Coverage

Question 18

How can using paired-end sequencing reduce the chance of introducing an error in the base calling?

Answer 18

We can check if there is a balanced calling of a genetic variant that is defected

Question 19

Where is the variant calling base balanced between in illumina paired-end sequencing?

Answer 19

Between read 1 and read 2 → otherwise the variation might be an error

Question 20

What are some pros of paired-end sequencing?

Answer 20

Millions of parallel sequencing reactions are performed and can lead to the identification of changes in the number of copies

Question 21

What are some cons of paired-end sequencing?

Answer 21

A very large amount of data is collected and both false positives and false negatives are possible

Question 22

Currently, what is required at the end of data analysis?

Answer 22

The use of Sanger sequencing because the results obtained by NGS require validation