Lecture 3

Question 1

What method was used to sequence the human genome?

Answer 1

Shotgun sequencing

Question 2

Is shotgun sequencing systematic or random?

Answer 2

Random

Question 3

How does shotgun sequencing work?

Answer 3

1. Genome is fragmented (sonication)
2. Fragments/smaller pieces are sequenced
   Fragments have an attached know DNA seq used as a marker
3. Find overlaps and assemble

Question 4

What is needed to find overlaps in sequences?

Answer 4

-Multiple copied of genome are needed
- Cut w/ staggered break points
-cannot have blunt ends

Question 5

What is a sequence library?

Answer 5

Collection of fragments

Question 6

Can you put the library into 1 tube to Sanger seq it?

Answer 6

No you would get mixed signals

Question 7

How are fragments isolated and copied in shotgun sequencing?

Answer 7

1. Fragment of interest is put into a vector
2. Electroporation →bacteria uptakes vector → inefficient most bacteria die, most don’t take up vector
3. Once inside bacteria, vector makes copies

Question 8

Even though electroporation is inefficient, why is it used to isolate fragments?

Answer 8

We rely on inefficiency of bacteria only getting 1 vector to isolate

Question 9

Whole genome sequencing

Answer 9

Take entire genome–> fragment them, sequence and find overlaps

Question 10

What method did public effort use to sequence human genome

Answer 10

Hierarchical Shotgun sequencing

Question 11

Steps of Hierarchical Shotgun sequencing

Answer 11

-Big random fragments
-Mapping
-Fragments were labeled where they came from
-Big fragments were shotgun sequenced
-Cloned into small vectors
-Sanger sequence
-Find overlaps

Question 12

What method did private effort use to sequence human genome

Answer 12

Whole genome shotgun sequencing

Question 13

Steps of Whole genome shotgun sequencing

Answer 13

-Small random fragments
-Cloned into small vectors
-Sanger sequence
-Find overlaps

Question 14

What is Illumina sequencing

Answer 14

Short reads sequencing millions of reads at a time

Question 15

What are the steps for Illumina Sequencing

Answer 15

1. Fragmentation
   -fragment randomly
   -attach adapters
2. Isolation and amplification
3. Sequencing by synthesis

Question 16

How to isolate fragments in Illumina seq?

Answer 16

1. Use flow cell → covered in DNA adapters that are complementary to adapters that are attached to fragments
2. Flow in dNTPs to make reverse complement strand
   Original template is washed away → so it does not move and start a new piece of DNA to be sequenced

Question 17

What kind of amplification is used in Illumina seq

Answer 17

Bridge Amplification
Make copies of bridge
Keep repeating until clusters are made (polonies) → PCR colonies

Question 18

What kind of dNTPs are used in Illumina sequencing by synthesis?

Answer 18

Reversible terminator fluorescent dNTPs
Temporary block on 3’ OH
No regular dNTPs are used

Question 19

Sequence by synthesis steps in Illumina

Answer 19

1. Flow in primer that is complementary to adapter
2. Reversible terminator fluorescent dNTPs
3. If complementary it will add
4. Take a pic
5. Remove terminator and fluorophore so only base is left
6. Repeat

Question 20

How to read Illumina sequence?

Answer 20

-Synthesized strand: Read from first photo to last photo or (bottom to top)
-Template strand: Read from last photo first photo while doing reverse complement at the same time (top to bottom)