Lecture 4 Strobel - Sequencing Genomes

Question 1

Define a genome.

Answer 1

the complete set of genes present in a cell or organism

Question 2

What is a transcriptome?

Answer 2

The complete set of coding and non-coding RNA transcripts in a cell, population of cells, or organism

Question 3

What is a proteome?

Answer 3

the complete set of proteins that are expressed by a cell, population of cells, or organism

Question 4

What was the goal of the Human Genome Project when it was launched in 1990?

Answer 4

they had a goal of determining the complete sequencing of the human genome

Question 5

What does the term “High-throughput sequencing” refer to?

Answer 5

its a general term that refers to any of several technologies that can sequence DNA in a rapid and cost effective manner.
* eg. Illumina/Solexa

Question 6

What is the goal of high-throughput DNA sequencing?

Answer 6

to determine the sequence of millions to billions of DNA sequences simultaneously

Question 7

Illumina sequencing sequences by what?

Answer 7

synthesis

Question 8

Explain what happens during Illumina sequencing.

Answer 8

1. nucleotides are attached to fluorescent dyes (different dyes for each base)
2. synthesize a new DNA strand one nucleotide at a time and read the sequence based on fluorescence

Question 9

What are the advantages of sequencing by synthesis (Illumina sequencing)?

Answer 9

1. it can sequence billions of different DNA molecules simultaneously
2. there is high accuracy (>99%)

Question 10

What are the disadvantages of sequencing by synthesis (Illumina sequencing)?

Answer 10

1. it requires expensive equipment
2. can only be used for short DNA sequences (typically 300 bp)
3. can only be directly applied to DNA

Question 11

What is primer extention?

Answer 11

a technique for in vitro DNA synthesis in which a primer is annealed to DNA and then extended by a DNA polymerase

Question 12

What is a chain terminator?

Answer 12

a nucleotide that, when incorporated into DNA or RNA, blocks further extension of the nucleic acid chain

Question 13

What does a reversible chain terminator do?

Answer 13

terminate primer extension reversibly

Question 14

After a reversible chain terminator is incorporated into DNA, what happens to DNA polymerase?

Answer 14

the DNA polymerase cannot extend the DNA chain further due to a blocking group on the 3’ OH

Question 15

What happens when you remove the blocking group on the 3’ OH?

Answer 15

removing the blocking group restores the 3’ OH
* once the 3’ OH is restored, DNA synthesis can proceed

Question 16

Explain what occurs during step 1 of sequencing of a genome using the Illumina platform.

Answer 16

Step 1: Prepare the genomic DNA sample
* Illumina can only accommodate short DNA fragment so you have to break the genomic DNA into small pieces

Question 17

What does Illumina sequencing require in order for it to work?

Answer 17

adapter sequences that are attached to the ends of the fragmented genomic DNA

Question 18

Explain what occurs during step 2 of sequencing of a genome using the Illumina platform.

Answer 18

Step 2: attach the DNA to the surface
* the DNA library is denatured (made single stranded) and attached to primers on a glass surface
* the primers are complementary to sequences in the adapters

Question 19

What is the significance of attaching DNA to a glass surface?

Answer 19

this helps the DNA to spread out different DNA molecules

Question 20

what occurs during step 3 of sequencing of a genome using the Illumina platform.

Answer 20

Bridge Amplification
* DNA bridges over to another primer on the glass surface

Question 21

what occurs during step 4 of sequencing of a genome using the Illumina platform.

Answer 21

DNA polymerase synthesizes a complementary DNA strand and the fragments become double-stranded

Question 22

what occurs during step 5 of sequencing of a genome using the Illumina platform.

Answer 22

the DNA has been duplicated and the process is repeated

Question 23

What does Illumina sequencing result in?

Answer 23

clusters of DNA molecules that are derived from the original genomic library

Question 24

What occurs during nanopore DNA sequencing?

Answer 24

1. DNA is threaded through a nanopore
2. DNA sequence is read as changes in current on either side of the pore

Question 25

What are the advantages of nanopore DNA sequencing?

Answer 25

1. long reads
2. can sequence DNA or RNA directly without significant biochemical processing
3. instruments are relatively inexpensive and portable

Question 26

What are the disadvantages of nanopore DNA sequencing?

Answer 26

1. less accurate than sequencing by synthesis (Illumina)
2. lower per-read throughput than sequencing by synthesis

Question 27

What threads the single stranded DNA through the nanopore that’s embedded in the membrane?

Answer 27

a helicase

Question 28

How is it possible to read a DNA sequence during nanopore sequencing?

Answer 28

by reading the flow of the ionic current through the nanopore