Module 6.1 Third Generation Sequencing Flashcards
1
Q
Third Generation Sequencing
features (6)
A
- 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
2
Q
Helicos Bioscience
features
A
- 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
3
Q
Helicos Bioscience
process
A
- fragment original DNA molecule
- melt into single stranded DNA
- add Poly A tail to DNA molecules through terminal transferase
- capture billions of DNA molecules on flow cell surface through base pairing with poly T oligos covalently and randomly anchored to glass cover slips
- add virtual terminators
- wash away unused nucleotides
- illuminate nucleotides with laser and detect signal with heliscope single molecule sequencer. Each spot on image is single nucleotide
- remove fluorescent signal before next nucleotide is added to continue cycle
4
Q
PacBio
Single Molecule Real Time (SMRT)
Sequencing
features (3)
A
- 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
5
Q
SMRT sequencing
phospholinked nucleotides
A
- fluorophores attached to 5’ phosphate group instead of base
- Phosphodiester bond formation releases fluorophore from incorporated nucleotide
- generates natural unmodified DNA
6
Q
SMRT sequencing
Zero Mode Waveguide
(ZMW)
features
A
- 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
7
Q
Single Molecule Real Time (SMRT)
Sequencing
process (6)
A
- bind single molecule of DNA template to Phi29 polymerase
- immobilize Phi29 at the bottom of ZMW
- illuminate fluorophore from below by laser light
- phospholinked nucleotide forms cognate association with DNA in polymerase active site -> elevates fluorescence output
- forms phosphodiester bond that releases dye linker pyrophosphate product
- polymerase translocates to next position and next cognate nucleotide binds active site for next pulse
8
Q
SMRT sequencing
HIFI reads
features (4)
A
- 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
9
Q
HIFI reads
library preparation
features (3)
A
- 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)
10
Q
Oxford Nanopore Technology
Nanopore sequencing
features (10)
A
- 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
11
Q
Nanopore sequencing
process (7)
A
- nanopore inserted into electrical resistance membrane surrounded by buffer electrolyte solution (eg. potassium chloride) split into two chambers
- voltage applied across membrane induces charged particle (ions in buffer solution) to pass through nanopore (very clean signal)
- motor protein on nanopore unwinds DNA strand ala helicase and provides passage for single strand DNA to enter nanopore channel
- negative molecular charge causes DNA to drift towards positive electrode and through nanopore
- nucleotide base interacts with and disrupts ionic current and recorded by patch clamp amplifier
- fingerprint mapped back to strand length and characteristics of component bases
- motor protein detaches after DNA/RNA passes through nanopore, ready for next fragment
12
Q
Oxford Technologies
Nanopore types
A
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
13
Q
Nanopore Sequencig
Library preparation
features (6)
A
- 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
