Module 6.1 Third Generation Sequencing

Question 1

Third Generation Sequencing

features (6)

Answer 1

• 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

Question 2

Helicos Bioscience

features

Answer 2

• first version of 3rd gen sequencing
• used a 3’ unblocked reversible terminator (virtual terminator)
• claimed to generate > 30 gigabases
• very short read length (32-35 bp).
• relatively high error rates
• relatively slow and expensive sequencing
• lost patent lawsuit to Illumina and incorporated into Direct Genomics

Question 3

Helicos Bioscience

process

Answer 3

1. fragment original DNA molecule
2. melt into single stranded DNA
3. add Poly A tail to DNA molecules through terminal transferase
4. capture billions of DNA molecules on flow cell surface through base pairing with poly T oligos covalently and randomly anchored to glass cover slips
5. add virtual terminators
6. wash away unused nucleotides
7. illuminate nucleotides with laser and detect signal with heliscope single molecule sequencer. Each spot on image is single nucleotide
8. remove fluorescent signal before next nucleotide is added to continue cycle

Question 4

PacBio

Single Molecule Real Time (SMRT)
Sequencing

features (3)

Answer 4

• directly observes processive DNA polymerization at base pair resolution in real time
• uses phospholinked nucleotide instead of base-linked fluorophore
• engineered Phi29 DNA polymerase incorporates phospholinked dNTPs very efficiently

Question 5

SMRT sequencing

phospholinked nucleotides

Answer 5

• fluorophores attached to 5’ phosphate group instead of base
• Phosphodiester bond formation releases fluorophore from incorporated nucleotide
• generates natural unmodified DNA

Question 6

SMRT sequencing

Zero Mode Waveguide
(ZMW)

features

Answer 6

• 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

Question 7

Single Molecule Real Time (SMRT)
Sequencing

process (6)

Answer 7

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

Question 8

SMRT sequencing

HIFI reads

features (4)

Answer 8

• 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

Question 9

HIFI reads

library preparation

features (3)

Answer 9

• 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)

Question 10

Oxford Nanopore Technology

Nanopore sequencing

features (10)

Answer 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

Question 11

Nanopore sequencing

process (7)

Answer 11

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

Question 12

Oxford Technologies

Nanopore types

Answer 12

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

Question 13

Nanopore Sequencig

Library preparation

features (6)

Answer 13

• 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