Week 2.3: Developments in Sequencing Technologies Flashcards
(39 cards)
1
Q
DNA Polymerase
Pyrophosphate
A
the two extra phosphate groups on a nucleoside triphosphate (eg. dCTP) used by DNA polymerase as an energy source to bond nucleotide to growing chain
2
Q
Second Generation Sequencing
Features
A
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
3
Q
NGS sequencing
Main steps
3
A
- Library preparation
- Immobilization
- Sequencing
4
Q
NGS sequencing
Immobilization
A
- 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
5
Q
NGS sequencing
Sequencing
A
- massive parallel cyclic sequencing reactions are performed to interrogate nucleotide sequence
- data analysis is carried out by computer
6
Q
Sequencing By Synthesis (SBS)
A
- 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
7
Q
Pyrosequencing
features (7)
A
- 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
8
Q
Pyrosequencing light detection
chemical reaction
A
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
9
Q
pyrosequencing
luciferase assay
features
A
- 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
10
Q
biotin
A
- vitamin B7or vitamin H.
- small water soluble compound
- high affinity for streptavidin
11
Q
streptavidin
A
- 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
12
Q
Emulsion PCR
A
- single stranded DNA fragments mixed with oligo-coated beads and PCR reagents (buffers, enzymes, primers)
- 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)
- micro beads surface is coated with hundreds of thousands of oligos complementary to B adapter.
- Oligo attached to beads through 5’ end
- DNA fragment anneals to oligo on bead surface
- The free 3’ end of oligo on the beads serves as PCR primer
- Another oligo which matches A adapter to serve as reverse primer for PCR is in the PCR solution
- The emulsion is then subject to PCR conditions,
- When PCR is complete, amplification products are denatured so that beads are covered with single stranded DNA (hairy capture bead)
13
Q
oligonucleotide
A
- short single-stranded or double-stranded fragments of DNA or RNA
- usually 13-25bp long, <200bp
14
Q
NGS sequencing
Bead enrichment procedure
A
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
15
Q
Pyrosequencing
Picotiter plate sequencing
process
A
- Beads randomly loaded on picotiter plate, one per 44-micron well (up to one million)
- layer of enzyme sequencing beads with sulfurylase and luciferase are added to ensure DNA beads remain in wells during sequencing
- In sequencer, nucleotides sequentially flowed across plate for hundreds of cycles
- polymerase extends existing DNA strand by adding the nucleotide to the 3’ end of primer
- Addition of one or more nucleotides will generate light signal that can be recorded by CCD camera
- Each light emission and its intensity interpreted by the computer to create a flow gram
- Signal strength proportional to homopolymer
16
Q
3’ blocked reversible terminators
A
- reversible blocking group is linked to Oxygen atom at 3’ carbon position of sugar ring (-OR instead of -OH)
- fluorescence label linked to base through cleavable linker
- terminator directly blocks 3’ hydroxy group so it has better termination effect
17
Q
3’ unblocked reversible terminator
A
- linked to base through cleavable linker
- fluorescent group functions as reporter AND as part of reversible terminating group
- relies on the fluorescent group to block 3’ hydroxy group
- less efficient than 3’ blocked reversible terminator
- easier to be accepted by DNA polymerase
18
Q
reversible terminating sequencing
Overview
A
- template and primer duplex are first immobilized on a solid support with DNA polymerase and the four reversible dye terminator nucleotides.
- primer extends strand by one base and stops
- wash away unincorporated nucleotides
- read and record color of fluorophore carried by extended base and identify incorporated nucleotide
- fluorescent tag and 3’ hydroxy blocking group are removed.
- after washing cycle, now have a primer template duplex with one base added to 3’ end of primer
- repeat steps to go through extension cleavage cycle
extension, termination, cleavage, extension
19
Q
reversible terminator limitations
molecular scar
A
- reversible terminator nucleotide analogs leave behind chemical scar after cleavage of linker carrying fluorescence
- accumulation snowballs, impairing stability of DNA double helix structure and hindering substrate recognition and primer extension
- contributor to short read lengths on Illumina sequencing platform
20
Q
NGS library prep method #1
adapter ligation
steps (4)
A
- fragmentation (100-400bp, <600)
- end repair
- phosphorylation
- A-tailing
21
Q
NGS sequencing adapter functions
3
A
- primer binding site for clonal PCR amplification
- sequencing primer binding
- sample indexing
22
Q
Adapter ligation
Sequencing adapter
features
A
- Y-shaped, 50bp in size
- 12 base pairs form double stranded stem through base pairing
- 5’ stem end is phophorylated
- 3’ stem end has single T base overhang
- short stem holds two single-strand of adapters together and enables ligation to double-stranded DNA insert
- T base overhang minimize chance of forming adaptor dimers without insert and can base pair with 3’ A-tail
- P5 and P7 primer binding sites for clonal amplification
- stretch of index sequences (unique sample barcodes) so you can put multiple samples together for sequencing and assign reads to each sample based on sample barcode
- Read 1 and Read 2 sequencing primers (Rd1/Rd2 SP) allows for pair end sequencing on sequencer
23
Q
NGS amplification
Bridging PCR
process
A
- one end of denatured library molecule anneals to oligo on flow cell complementary to 3’ adapter
- polymerase extends from 3’ end of the oligo to make full copy of library molecule
- double stranded DNA is denatured and original template washed away.
- Attached strand bends over and 3’ end anneals to oligo complementary to other adapter
- DNA polymerase makes another copy
- DNA is denatured and now have two copies of original molecule attached to flow cell
24
Q
NGS amplification
Flow cell clusters
A
- millions of clusters generated with thousands of copies of library molecules on flow cell
- ensures enough fluorescent signal is emitted
- DNA strands attached to P5 (read 2) or P7 (read 1) removed by specific base cleavage to leave identical sequences
- 3’ end of DNA strand and flow cell-bound oligos are blocked to prevent interference with sequencing reaction
25
# NGS
Read 1 Sequencing
| process (9)
1. Read 1 sequencing primer anneals to primer binding site
2. All four nucleotides, each with different fluorescent label, and polymerase are added to flow cell
3. polymerase incorporates first matched nucleotide fluorescently labeled with 3' end blocked by reversible terminator
4. extra nucleotides are washed away and four pictures taken, one for each color, capturing all fluorescent signal from flow cell after first base incorporation
5. chemical added to remove fluorescent and blocking groups
6. process repeats
7. each cluster is at specific location on flow cell and generates one read
8. fluorescent signal data analyzed to infer DNA sequence within each cluster
9. index primer is annealed to template and reads index 1 (8bp) at end of DNA
26
# NGS
Read 2 Sequencing
| process
1. molecules denatured to remove newly synthesized DNA strand
2. molecules on flow cell undergo bridging PCR
3. remove strand attached through oligo complementary to P7
4. Read 2 primer anneals to template
5. Repeat SBS process
6. Index 2 barcode may be sequenced before or after Read 2 bridge amplification
27
demultiplexing
| process
- computational process where sequencing reads from pooled libraries need to be identified, sorted, and assigned to their respective samples based on indexes incorporated during library preparation
- crucial in multi sample sequencing experiments, where multiple libraries are pooled and sequenced together in a single sequencing
28
index hopping
-happens when a cluster with a particular barcode or index is misread as a different barcode during sequencing process
- reads originally associated with one sample incorrectly assigned to different sample
- creates cross-contaminated libraries
- more severe in pattern flow cells
Causes
- contamination from free adapters not fully removed during magnetic bead purification. remaining free adapters can function as PCR primers
- Pattern flow cell ExAmp has more free adapters pooled with samples
- avoid by us unique dual index strategies and remove free adapters after ligation
29
# reversible terminator limitations
phasing
- contribute to errors in base calling
- cause the strands within a cluster to be out of sync for base incorporation during each cycle and generate mixed fluorescent signals and will affect the quality score of the read
- accumulation of phasing and prephasing events within cluster will lead to significant drop of the read quality in later cycles towards end of read
30
Third Generation Sequencing
| features (6)
- aka single molecule sequencing
- no clonal amplification
- SMRT (PacBio) and Nanopore (Oxford Technologies)
- weaker signals = lower accuracy
- can directly detect epigenetic markers
- useful for genome assembly, structural variant detection, and understanding complex genomic landscapes
31
# SMRT sequencing
phospholinked nucleotides
- fluorophores attached to 5’ phosphate group instead of base
- Phosphodiester bond formation releases fluorophore from incorporated nucleotide
- generates natural unmodified DNA
32
# SMRT sequencing
Zero Mode Waveguide
(ZMW)
| features
- nanophotonic confinement structure used to observe single molecule incorporation
- circular holes 70 nm wide and 100 nm deep
- small confinement volume can detect single nucleotide incorporation despite high concentration of labeled dNTP in bulk solution
33
Single Molecule Real Time (SMRT)
Sequencing
| process (6)
1. bind single molecule of DNA template to Phi29 polymerase
2. immobilize Phi29 at the bottom of ZMW
3. illuminate fluorophore from below by laser light
4. phospholinked nucleotide forms cognate association with DNA in polymerase active site -> elevates fluorescence output
5. forms phosphodiester bond that releases dye linker pyrophosphate product
6. polymerase translocates to next position and next cognate nucleotide binds active site for next pulse
34
# SMRT sequencing
HIFI reads
| features (4)
- Phi29 SDA activity enables closed circular template to be sequenced multiple times by polymerase in single run
- can compare sequence and remove inconsistent errors between sub reads
- >99% accurate for >1000 bases
- can detect DNA synthesis kinetics in real time
35
# HIFI reads
library preparation
| features (3)
- SMRTbell hairpin adapters ligate on DNA fragment ends to create circular template
- sequencing primers and polymerase bind at loop sequence in adapter to initiate sequencing
- can create libraries of various insert sizes (250-25,000bp)
36
# Oxford Nanopore Technology
Nanopore sequencing
| features (10)
- utilize nanoscale pores (nanopores) to detect nucleic acid sequences
- nucleotide fingerprint based on event duration and magnitude of current blockage
- ATP dependent molecular motors
- user can control fragment lengths during the library prep
- read length = molecule length
- can read over 4 megabases (4 million bases)
- 6-10% error rate
- high portability, low cost, and ease of use for rapid sequencing.
- useful for spanning repeats
37
Nanopore sequencing
| process (7)
1. nanopore inserted into electrical resistance membrane surrounded by buffer electrolyte solution (eg. potassium chloride) split into two chambers
2. voltage applied across membrane induces charged particle (ions in buffer solution) to pass through nanopore (very clean signal)
3. motor protein on nanopore unwinds DNA strand ala helicase and provides passage for single strand DNA to enter nanopore channel
4. negative molecular charge causes DNA to drift towards positive electrode and through nanopore
5. nucleotide base interacts with and disrupts ionic current and recorded by patch clamp amplifier
6. fingerprint mapped back to strand length and characteristics of component bases
7. motor protein detaches after DNA/RNA passes through nanopore, ready for next fragment
38
# Oxford Technologies
Nanopore types
**biological nanopores**: protein nanopores embedded in lipid membranes that create size-dependent porous surfaces
**solid state nanopores**: use various metal / alloy substrates with nanometer size pores
39
# Nanopore Sequencig
Library preparation
| features (6)
- optional DNA fragmentation and size selection
- end repair and A-tailing
- sequencing Y-adapters with motor proteins on 5' ends ligated onto repaired ends
- motor protein controls translocation of DNA or RNA strand through nanopore
- pairing of nanopore to motor proteins like Phi29 DNA polymerase or helicase produce slow translocation -> more sensitive current alterations = single based discrimination
- also offers transposase workflow like Nextera library to add adapters
