DNA Sequencing

Question 1

Minimum requirements for DNA synthesis in vitro

Answer 1

• most methods of DNA sequencing are based on DNA synthesis

- DNA synthesis proceeds in 3’ direction

Question 2

Formation of phosphodiester bond

Answer 2

• catalyzed by DNA polymerase
• nucleophilic attack on the 3’-OH on the innermost phosphorous atom of the incoming dNTP
• dideoxyriboucleoside triphosphate (ddNTP) terminates DNA synthesis (because the 3’-OH is non-existent)
• when dd(G)TP is incorporated instead of dGTP there is no further extension of the strand
  
  • this gives DNA daughter strands of varying length

Question 3

Spiking DNA

Answer 3

• could spike the DNA polymerization cocktail with small amounts of ddATP, ddCTP, ddTTP, ddGTP
• in this case we would get a subset of DNA elongation products terminating with a ddNTP base at every position in the DNA sequence
• to keep track of which bases are terminating we attach different fluorescent colours to each type of ddNTP so that we can see which colour/ddNTP comes next in sequence
• to sort the fragments by size (to identify correct order) we use gel electrophoresis

Question 4

Fluorescent dideoxy sequencing

Answer 4

• usually automated
• gel electrophoresis uses denaturing polyacrylamide gel (contains urea) to separate fragments by size
• this type of gel gives very fine resolution, ability to distinguish fragments that differ by 1 base in size
• as ddNTP-terminated fragments migrate in the gel, they pass a laser beam that excites the fluorescent dyes and a CCD camera that records the flash of coloured light that results
• software converts raw data to electropherogram and DNA sequence

Question 5

Before fluorescent sequencing…

Answer 5

• before fluorescent sequencing technology was invented, radioactive labeling was used to detect bands on DNA sequencing gels
• 4 separate sequencing reactions, each containing a different ddNTP
• primers labelled radioactively
• x-ray film was used to prepare autoradiograph

Question 6

Sanger dideoxy sequencing pros

Answer 6

• very accurate (low rate of sequencing error)
• relatively long sequencing reads (up to 1000b but 650b more common)
• easy and can be automated
• low cost (for small number of samples)

Question 7

Sanger dideoxy sequencing cons

Answer 7

• too slow for many applications, such as genome sequencing
• costly when scaled up to acquire lots of data
• requires purification and preparation of each individual DNA sequence that is being studied
• these limitations led to invention of next generation methods

Question 8

Sequencing capacity then and now

Answer 8

-Human genome project consortium in 2000: 8.64x10^7 bases per day

• Marine gene probe lab dalhousie in 2015:
  1. 5x10^10 bases per day

-human genome project completed using dideoxy sequencing and took 10 years and 3billion$

Question 9

Pyrosequencing

Answer 9

• developed by Pal Nyren and Mostafa Ronaghi at Royal institute of technology in 1996
• now commonly known as 454 sequencing
• depends on detection of pyrophosphates when dNTPs are added to growing DNA chain

Question 10

Pyrosequencing step 1

Answer 10

• a sequencing primer hybridized to a single stranded DNA
• strand extension occurs in presence of DNA polymerase, ATP sulfurylase, luciferase, and apyrase, adenosine 5’ phosphosulfate (APS) and luciferin

Question 11

Pyrosequencing step 2

Answer 11

• 1st dNTP is added to the reaction
• DNA polymerase catalyzes incorporation of dNTP into growing DNA strand IF it is complementary to the base in the DNA template strand
• each incorporation even = release of pyrophosphate (PPi) in a quantity equimolar to the amount of the incorporated nucleotide

Question 12

Pyrosequencing step 3

Answer 12

• ATP sulfurylase converts PPi + APS —> ATP
• ATP + luciferase + luciferin —> oxyluciferin + visible light (in proportion to amount of ATP)
• light is detected by a camera and seen as a peak in the raw data output (pyrogram)
• height of each peak is proportional to the number of nucleotides incorporated

Question 13

Pyrosequencing step 4

Answer 13

• Apyrase degrades unincorporated nucleotides and ATP

- when degradation is complete another nucleotide is added

Question 14

Pyrosequencing step 5

Answer 14

• addition of dNTPs performed sequentially
• as the process continues, the complementary DNA strand is built up and the nucleotide sequence is determine from the signal peaks i the pyrogram trace

Question 15

Semiconductor sequencing

Answer 15

• Ion torrent
• similar to pyrosequencing but instead of pyrophosphate, H+ ion is detected
• like pyrosequencing, begins with emulsion PCR
• DNA templates are on microscopic beads
• sequencing occurs on a modified computer chip
• hydrogen ions are detected in a layer of chip below bead wells (worlds smallest pH meter)
• no modified chemistry
• no camera needed
• very fast (a couple of hours)

Question 16

Illumina DNA sequencing

Answer 16

1. Prepare genomic DNA sample
   
   • randomly fragment genomic DNA and legate adapters to both ends of the fragments
2. Attach DNA to surface
   
   • bind single stranded fragments randomly to inside surface of the flow cell channels
3. Bridge amplification
   
   • add unlabelled nucleotides and enzyme to initially solid-phase bridge amplification
4. Fragments become double stranded
   
   • the enzyme incorporates nucleotides to build double-stranded bridges on the solid-phase substrate
5. Denature the double stranded molecules
   
   • leaving single stranded templates anchored to the substrate
6. Complete amplification
   
   • several million dense clusters of double stranded DNA are generated in each channel of the flow cell

Question 17

Illumina DNA sequencing Part 2

Answer 17

7. Determine first base
   
   • to initiate the sequencing cycle, add all four labeled reversible terminators, primers, and DNA polymerase enzyme to the flow cell
8. Image first base
   
   • after laser excitation, capture the image of emitted fluorescence from each cluster on the flow cell. Record the identity of the first base of each cluster
9. Determine 2nd base
   
   • repeat step 7
10. Image 2nd chemistry cycle
    - repeat step 8
11. Sequence reads over multiple chemistry cycles
    - repeat cycles of sequencing to determine the sequence of bases in a given fragments a single base at a time
12. Align data
    - align data and compare to a reference and identify sequence differences

Question 18

Performance/cost comparisons of sequencing instruments

Answer 18

Most expensive per MB:

1. dideoxy ($2308)
2. 454 FLX titanium ($12) - pyrosequencing
3. Ion torrent ($7)
4. Illumina MiSeq ($0.20)
5. Illumina HiSeq 1000 ($0.04)

Question 19

3rd gen: nanopore sequencing

Answer 19

• single molecule at a time (no pre amplification by PCR)
• enzyme unwinds DNA; a single strand is pulled by an electrical current through a pore in a membrane
• each base produced a characteristic disturbance in electrical current which can be used to read the bases as it travels through the pore

Question 20

Nanopore pros and cons

Answer 20

• pros:
  
  • long reads (up to 100kb)
  • no PCR step
  • small, highly portable DNA sequencer connects to USB port
  • can be used in the field
• cons:
  
  • low accuracy compared to other methods but getting better

Question 21

3rd gen: PacBio

Answer 21

• older single molecule DNA sequencing method than Nanopore
• reads lengths 20-60kn
• not as accurate as illumina or ion torrent
• often used in combo with illumina when sequencing genomes