Module 3.2 Next Generation Sequencing I

Question 1

Second Generation Sequencing

Features

Answer 1

massive parallel sequencing
- clonal amplification of DNA molecule where millions or billions of different DNA fragments get sequenced at the same time in parallel fashion and generate enormous data
happens on a solid surface (beads or glass slide)
- doesn’t require the physical separation of reactions in different wells or tubes
capacity to produce massive volume of data from a single run at a very low cost and in a short duration without bacterial cloning generally used in Sanger sequencing

much lower labor input and cost compared to first gen sequencing

Question 2

NGS sequencing

Main steps

3

Answer 2

1. Library preparation
2. Immobilization
3. Sequencing

Question 3

NGS sequencing

Library Preparation

process (5)

Answer 3

1. DNA cut into 300-700bp fragments
2. blunt end repaired and ligated with universal adapters
3. Mixed with streptavidin-beads
4. AA adapters washed away
5. Denature DNA w/ alkaline treatment, releases 5’A-3’B strand into solution for clonal amplification

Question 4

NGS sequencing

Immobilization

Answer 4

• prepared sequencing library fragments are immobilized on a solid surface and amplified to form detectable sequencing features.
• Each feature/spot on solid support corresponds to one original DNA fragment.
• each fragment will lead to a read or a pair of reads which is equivalent to one capillary of gel electrophoresis of sanger sequencing
• ensures sufficient signal for detection

Question 5

NGS sequencing

Sequencing

Answer 5

• massive parallel cyclic sequencing reactions are performed to interrogate nucleotide sequence
• data analysis is carried out by computer

Question 6

Sequencing By Synthesis (SBS)

Answer 6

• rely on the principle of synthesizing a complementary strand of DNA through DNA replication.
• determines sequencing of a template by detecting incorporation of a nucleotide through DNA polymerase

Question 7

Pyrosequencing

features (7)

Answer 7

• aka 454 Sequencing (Roche)
• 1st developed SBS NGS technology
• detects DNA synthesis byproduct pyrophosphate in real time
• Long read length: up to 700bp
• Throughput: up to 1 million reads per run
• Accuracy: high homopolymer errors
• Cost: cheaper than Sanger sequencing, but relatively high compared to other NGS systems

Question 8

Pyrosequencing light detection

chemical reaction

Answer 8

1st reaction
- ATP sulfurylase (ATP sulfate adenylyltransferse) converts pyrophosphate and adenylyl sulfate to ATP and sulfate (reversible)

2nd reaction
- firefly luciferase catalyzes oxidization of firefly luciferin
- forms oxyluciferin in electronically excited state
- releases photon of light as oxyluciferin goes back to ground state
- requires oxygen and ATP
- 2nd reaction utilizes ATP generated by 1st reaction

Question 9

pyrosequencing

luciferase assay

features

Answer 9

• can measure a stable level of light produced in the reaction
• light emission is proportional to the ATP concentration.
• can be completed in less than 2 seconds and not affected by inorganic phosphate.
• extremely sensitive with a linear range of 10^-9 to 10^-7 molar pyrophosphate
• suitable for continuous real time monitoring of pyrophosphate formation at extremely low amount

Question 10

Pyrosequencing

solid phase column

process (8)

Answer 10

1. adenylyl sulfate, glucose, glycerol, luciferin and one of the four DNTP’s pumped through capillary column
2. pyrophosphatase to remove residual pyrophosphate
3. template primer DNA and polymerase
4. remove any trace amount of ATP or dATP
5. ATP sulfurylase to catalyze converting pyrophosphate to ATP
6. firefly luciferase column to catalyze luciferin + ATP = luciferin oxidation + light
7. light emitted is detected by a photomultiplier tube
8. wash column buffers and introduce different dNTP

Question 11

Pyrosequencing

dATPaS

Answer 11

-analog of ATP
- reduces background noise introduced by nonspecific interactions between ATP and luciferase

Question 12

NGS sequencing

A & B universal adapters

Answer 12

short synthetic double strand DNA molecules that differ in sequence

contain 44 base pairs
- 20 for PCR amplification,
- 20 for sequencing primer binding,
- 4 as sample index

three purposes
- clonal amplification on the solid surface.
- sequencing.
- sample indexing.

blunt on one end and recessed on other end
B adaptor carries a 5’ biotin at 5’ end (biotinylated oligo)

Question 13

biotin

Answer 13

• vitamin B7or vitamin H.
• small water soluble compound
• high affinity for streptavidin

Question 14

streptavidin

Answer 14

• protein in the form of a tetramer.
• Each unit of the tetramer can accommodate one biotin molecule.
• biotin + streptavidin = one of the strongest non covalent interactions in nature

Question 15

Emulsion PCR

Answer 15

1. single stranded DNA fragments mixed with oligo-coated beads and PCR reagents (buffers, enzymes, primers)
2. mixture transferred to tube containing oil and shaken to create water droplets in oil with one bead, one DNA fragment and PCR reagents (excess beads)
3. micro beads surface is coated with hundreds of thousands of oligos complementary to B adapter.
4. Oligo attached to beads through 5’ end
5. DNA fragment anneals to oligo on bead surface
6. The free 3’ end of oligo on the beads serves as PCR primer
7. Another oligo which matches A adapter to serve as reverse primer for PCR is in the PCR solution
8. The emulsion is then subject to PCR conditions,
9. When PCR is complete, amplification products are denatured so that beads are covered with single stranded DNA (hairy capture bead)

Question 16

oligonucleotide

Answer 16

• short single-stranded or double-stranded fragments of DNA or RNA
• usually 13-25bp long, <200bp

Question 17

NGS sequencing

Bead enrichment procedure

Answer 17

Remove beads without amplified DNA after emulsion PCR

• mix the beads with biotinylated primer B that hybridizes to the single stranded DNA copies on bead surface.
• use beads coated with streptavidin to select for beads with primer B with biotin
• beads with clonally amplified DNA fragments can then be recovered with magnet

Question 18

Pyrosequencing

Picotiter plate sequencing

process

Answer 18

1. Beads randomly loaded on picotiter plate, one per 44-micron well (up to one million)
2. layer of enzyme sequencing beads with sulfurylase and luciferase are added to ensure DNA beads remain in wells during sequencing
3. In sequencer, nucleotides sequentially flowed across plate for hundreds of cycles
4. polymerase extends existing DNA strand by adding the nucleotide to the 3’ end of primer
5. Addition of one or more nucleotides will generate light signal that can be recorded by CCD camera
6. Each light emission and its intensity interpreted by the computer to create a flow gram
7. Signal strength proportional to homopolymer

Question 19

Ion semiconductor sequencing

sequencing detection process

Answer 19

• ion sensitive layer and ion sensor under microwell
• All layers contained within semiconductor chip that contains an array of microvalves with corresponding detectors
• Protons are released when nucleotides are incorporated on the growing DNA strand, changing the pH of well
• changes surface potential of metal oxide sensing layer and source terminal potential of underlying ion sensor
• series of electrical pulses transmitted from chip to computer is translated into DNA sequence with no intermediate signal conversion

Question 20

Ion semiconductor sequencing

Answer 20

• 200-600 base pair read lengths.
• throughput ranges 2-3 million to up to 130 million reads
• electronic insted of optic detection
• cheaper and faster than other NGS platforms