Lecture 3 Flashcards
What method was used to sequence the human genome?
Shotgun sequencing
Is shotgun sequencing systematic or random?
Random
How does shotgun sequencing work?
- Genome is fragmented (sonication)
- Fragments/smaller pieces are sequenced
Fragments have an attached know DNA seq used as a marker - Find overlaps and assemble
What is needed to find overlaps in sequences?
-Multiple copied of genome are needed
- Cut w/ staggered break points
-cannot have blunt ends
What is a sequence library?
Collection of fragments
Can you put the library into 1 tube to Sanger seq it?
No you would get mixed signals
How are fragments isolated and copied in shotgun sequencing?
- Fragment of interest is put into a vector
- Electroporation →bacteria uptakes vector → inefficient most bacteria die, most don’t take up vector
- Once inside bacteria, vector makes copies
Even though electroporation is inefficient, why is it used to isolate fragments?
We rely on inefficiency of bacteria only getting 1 vector to isolate
Whole genome sequencing
Take entire genome–> fragment them, sequence and find overlaps
What method did public effort use to sequence human genome
Hierarchical Shotgun sequencing
Steps of Hierarchical Shotgun sequencing
-Big random fragments
-Mapping
-Fragments were labeled where they came from
-Big fragments were shotgun sequenced
-Cloned into small vectors
-Sanger sequence
-Find overlaps
What method did private effort use to sequence human genome
Whole genome shotgun sequencing
Steps of Whole genome shotgun sequencing
-Small random fragments
-Cloned into small vectors
-Sanger sequence
-Find overlaps
What is Illumina sequencing
Short reads sequencing millions of reads at a time
What are the steps for Illumina Sequencing
- Fragmentation
-fragment randomly
-attach adapters - Isolation and amplification
- Sequencing by synthesis
How to isolate fragments in Illumina seq?
- Use flow cell → covered in DNA adapters that are complementary to adapters that are attached to fragments
- 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
What kind of amplification is used in Illumina seq
Bridge Amplification
Make copies of bridge
Keep repeating until clusters are made (polonies) → PCR colonies
What kind of dNTPs are used in Illumina sequencing by synthesis?
Reversible terminator fluorescent dNTPs
Temporary block on 3’ OH
No regular dNTPs are used
Sequence by synthesis steps in Illumina
- Flow in primer that is complementary to adapter
- Reversible terminator fluorescent dNTPs
- If complementary it will add
- Take a pic
- Remove terminator and fluorophore so only base is left
- Repeat
How to read Illumina sequence?
-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)
