Module 5.2 Emerging NGS Platforms

Question 1

Phi29 DNA Polymerase

Answer 1

• enzyme from bacteria phage Phi29
• simple monomer protein
• C-terminal domain: polymerase
• N-terminal domain: strong 3’ to 5’ exonuclease activity
  Exceptionally High processivity:
• capable of incorporating more than 10,000 nucleotides in a single binding event, one of the most processive known
  Strong proof reading activity:
• may produce 1-2 orders of magnitude less error than Taq polymerase
• Highly processive
• can degrade single stranded oligonucleotides without dissociation
  Remarkable Strand-displacement activity
• acts similar to helicase through strong binding to single stranded DNA.
• optimal temp = 30 degrees (isothermal)

Question 2

Strand displacement Amplification

Answer 2

ability to displace or push aside a downstream complementary DNA strand while synthesizing DNA using another strand as template in the 5’ to 3’ direction.

Question 3

Multiple Displacement Amplification
(MDA)

process

Answer 3

1. multiple random hexamer primers bind to DNA template
2. strand displacing polymerase (Phi29) amplifies from primer binding sites.
3. extension progresses in 5’ to 3’ direction
4. Phi29 displaces newly synthesized strand on 3’ side and creates single stranded branches that can bind new random hexamer primers
5. primers and polymerase continue to bind and amplify on branched strands
6. end results >30 KB and highly branched networks of DNA

Question 4

Rolling Circle Amplification
(RCA)

single primer process

Answer 4

• primer hybridizes to template at target DNA sequence
• strand-displacement DNA polymerase (eg. Phi29) extends primer along circle template and displaces newly synthesized strand to continues making new strand
• process continues in a rolling circle manner forming of a long single stranded DNA product composed of repeated units of original circular template.
• resembles a tandem array of the target sequence

Question 5

tandem array

Answer 5

A series of copies of a gene arranged one after another along a chromosome

Question 6

Rolling Circle Amplification
(RCA)

multiple primer process

Answer 6

• can use multiple primers (random or target-specific) that are complementary to forward and reverse strands
• primers amplify circular DNA in one direction and displace newly synthesized strand
• displaced single strands amplified by primers in other direction like MDA
• bidirectional amplification continues to produce long and highly branched double strand DNA with repeat units

Question 7

Complete Genomics

Library preparation

adapter features

Answer 7

• Read 1 and 2 adapters bind together to form D-loop structure
• Read 1= 5’ stem -> D loop -> T overhang 3’
• Read 2= 3’ overhang-> sample index -> stem -> phosphate group 5’

Question 8

Complete Genomics

Library Preparation

process

Answer 8

• target DNA fragmented to proper size and end-repaired to achieve the blunt end with 5’ phosphate group and A-tailing at 3’ end
• ligate adapters to ends of target molecule to create library molecules
• PCR forward primer matches loop, reverse primer matches 3’ overhang
• create the double stranded read 1 adapter, dna fragment, read 2 adapter with sample index

Question 9

DNA Nano Balls
(DNB)

process

Answer 9

1. library molecules denatured to create two single stranded molecules.
2. forward strand used to form circular DNA
3. single stranded library molecule annealed to splint molecule that brings two ends of library molecules together, forming short stretch of double strands with a nick.
4. nick is sealed by ligase to form a closed circle.
5. splint molecules and non ligated linear molecules removed using DNA exonuclease
6. single strand DNA circle replicated with Phi29 polymerase via RCA using primer that specifically binds to adapter sequence
7. Generates long single stranded DNA with hundreds of tandem copies of library molecule
8. Under proper buffer conditions, DNA folds into 3D ball about 220 nm in diameter in solution

Question 10

splint molecule

Answer 10

a single stranded, short sequence that is complementary to 5’ and 3’ end of adapter

Question 11

DNB

DNA exonuclease

Answer 11

Enzyme which specifically digests linear DNA without cutting circular DNA

Question 12

DNB

Rolling Circle Amplification
(RCA)

benefits and drawbacks

Answer 12

Benefits
- linear molecules removed by exonuclease minimize the index hopping risk
- no polymerase dissociation and reassociation and Phi29 high processivity minimizes risk of truncation during clonal amplification.
- higher fidelity amplification through continuous copying of the original circular DNA template. Avoids creation of clonal errors like in PCR

Drawbacks
- small insert fragment size (<700bp, 500bp preferred)
- longer inserts are harder to ligate to form a circle and fewer copies within DNA ball.
- Too few copies in DNA ball = weak signal. Copy number limited by insert size and reaction time

Question 13

DNB

Patterned Array

features

Answer 13

• solid surface with grade patterned arrays of positively charged spots fabricated via photolithography, etching techniques then chemical modification to generate a sequencing flow cell.
• One spot = 300 nm diameter,separated by 700 nm from center to center to allow easy attachment of DNB to flow cell.
• increases DNA content per array and image information density relative to non pattern arrays

Question 14

Cool MPS

features

Answer 14

• no fluorescently labeled reversibly terminated nucleotides required
• uses unlabeled nucleotides with reversible termination group at the 3’ carbon position, which will pause the DNA polymerase upon incorporation into growing chain
• base determination via fluorescently labeled antibodies

Question 15

Cool MPS

process

Answer 15

1. Sequencing primers bind to hundreds of adapters
2. DNA polymerase adds 1 of 4 unlabeled, reversibly terminating nucleotides to 3’ end of primer
3. unbound nucleotides are washed away, and DNA synthesis is paused by blocking group on 3’ end.
4. 4 types of fluorescent labeled base-specific monoclonal antibodies that only bind bases with blocking groups are added to flow cell
5. unbound antibodies are washed away
6. fluorescent signal captured by camera and converted to digital output for base calling
7. bound antibodies and blocking groups removed to regenerate natural nucleotides with no scar to allow longer extension
8. cycle repeated

Question 16

Cool MPS

monoclonal antibodies

Answer 16

lab-made proteins that bind to only one type of antigen

Question 17

Cool MPS

benefits and drawbacks

Answer 17

Benefits
- nucleotide incorporation and identification steps are decoupled
- unlabeled bases incorporated more efficiently
- no molecular scar
- unlabeled reversible terminators are easier and less costly to make.
- longer reads with higher quality.
- antibodies can carry multiple (2-5) molecules of same dye, providing stronger sequencing signal

Drawbacks
- high cost of set up
- once the antibody clones are produced, ongoing generation cost can be minimal
- more time required for antibody binding = slower sequencing

Question 18

Element BioSciences

Avidity Sequencing

features

Answer 18

• separate terminator and fluorescent detection reagents
• increased base calling accuracy and read lengths
• lower reagent cost relative to the combo terminator/dye labeled nucleotide
• uses 3’ blocked but unlabeled nucleotides to extend the sequencing primer chain
• uses constructed polymer instead of antibody for signal detection

Question 19

Avidity sequencing

Avidite

Answer 19

• constructed polymer
• protein core consists of fluorophore-labeled streptavidin
• three arms connected to three identical nucleotides (biotin, linker, nucleotide)
• one arm linked to additional cores
• structure allows it to form stable structure with DNA polymerase and DNA substrate during sequencing for fluorescent signal detection

Question 20

Avidity Sequencing

process

Answer 20

1. DNA fragments ligated with adapters (seq adapter & index)
2. library molecule is circularized through splint oligo and ligase
3. circularized DNA library molecules captured on flow cell through base pairing between adapter sequence on library molecule and surface primers that coat flow cell
4. Hundreds of clonal copies of DNA fragments created through RCA, generating concatemers on flow cell
5. DNA strand rolls into tight ball (polony), can generate about 1 billion on flow cell and
6. Mixture of four avidites (one for each base/fluorescent label), sequencing primers, and polymerase applied to flow cell
7. DNA polymerases bind to many free 3’ ends of template-hybridized primers of a polony and select correct cognate Avidite via base pairing
8. In presence of DNA polymerase, Avidite able to non-covalently bind multiple complementary nucleotides within polony that ensures a long signal persistence time
9. Unbound Avidite washed away, Fluorescent bound Avidite imaged
10. Avidites removed from polonies without modifications or molecular scars on synthesized strand
11. remove blocking group, cycle repeats
12. Multiple avidites bind to each polony

Question 21

Avidity sequencing

concatemer

Answer 21

• a long continuous DNA molecule that contains multiple copies of the same DNA sequence linked in series.
• usually copies of entire genome linked end to end and separated by cos sites (a protein binding nucleotide sequence that occurs once per genome copy)

Question 22

polony

Answer 22

• tiny colony of DNA (1 micron diameter) grown on a glass microscopic slide
• mashup of “polymerase colony”
• simplifies user workflow and eliminates DNA fragments from interacting in solution

Question 23

Avidity Sequencing

benefits

Answer 23

Benefits
- decouples nucleotide incorporation identfication
- synergistic binding of multiple nucleotides on a single avidite ensures only correct avidite binds to polony
- effectively remove errors caused by phasing or pre phasing
- retains good performance in reads containing long homopolymers

Question 24

Mostly Natural Sequencing By Synthesis (MNSBS)

features

Answer 24

• utilizes single base from a mostly natural non-terminated nucleotide mix (more unlabeled than fluorescently labeled)
• bases efficiently measured via high-throughput optical scanning
• fluorescent label produces hundreds of photons = higher signal:noise ratio = better detection
• cycle length = 2 min
• amount of labeled nucleotide incorporated on clonal beads linearly proportional to length of homopolymer, while each individual synthesized DNA strand remains mostly unlabeled
• uses circular 200 nm meter silicon wafer as open flow cell- reused within instrument
• wafer patterned at micron scale, creating dense array of electrostatic landing pads to bind clonally amplified sequencing beads

Question 25

Mostly Natural Sequencing By Synthesis (MNSBS) | process

Answer 25

1. emulsion PCR for clonal amplification 2. beads with clonally amplified library molecules uniformly distributed on sequencing wafer each cycle 3. template primer complex exposed to mostly natural nucleotide mix of a single base like C, A, G, or T 4. polymerase extension performed to incorporate 0+ bases of that single base type into each growing strand 5. entire active surface of sequencing wafer is scanned to measure the fluorescent level of each clonal bead 6. all fluorescent labels cleaved and washed before repeating cycle

Question 26

Mostly Natural Sequencing By Synthesis (MNSBS) | benefits and drawbacks

Answer 26

**Benefits** - has better signal:noise ratio with fluorescent signals instead of photons or protons that have high intrinsic noise - may potentially avoid sequencing of fluorescent signals from adjacent labels - can produce longer homopolymer reads (<12bp) than pyro and ion conductor - no molecular scars = faster run time and a longer read length (>400bp) - high active reagent utilization and lower reagent consumable cost - claims development of unique base calling algorithms that use machine learning and deep convolutional neural network for sequencing reads - claims high level of system robustness and more accurate base calls **Drawbacks** - homopolymer accuracy is still lower than other sequencing platform using reversible terminators - 10% error rate for 8-mer homopolymer

Question 27

# MNSBS Spin dispense system

Answer 27

- delivers sequencing reagents to wafer by dispensing reagents from dedicated nozzle near center of rotating wafer - distribute reagents rapidly and uniformly across wafer by centrifugal force, forming thin layer on surface - optical measurement of entire surface is performed during rotation of wafer in continuous process, like reading a compact disc

Question 28

Clonal Amplication Comparison

Answer 28

- **Bridging PCR/Recombinase Polymerase Amplification**: Illumina - **Emulsion PCR**: Ion Torrent & Ultima Genomics - **RCA**: Complete Genomics & Element Biosciences

Question 29

Platform Sequencing Chemistry

Answer 29

- **Illumina**: SBS with reversible dye terminator - **Ion Torrent**: SBS with proton detection - **Complete Genomics**: HotMPS(reversible dye terminator) / CoolMPS (Antibody) - **Element BioScience**: Avidity Sequencing - **Ultima Genomics**: mnSBS