Module 4.1 Next Generation Sequencing II Flashcards
3’ blocked reversible terminators
- 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
3’ unblocked reversible terminator
- 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
reversible terminating sequencing
Overview
- 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
reversible terminator limitations
molecular scar
- 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
NGS library prep method #1
adapter ligation
steps (4)
- fragmentation (100-400bp, <600)
- end repair
- phosphorylation
- A-tailing
adapter ligation
physical disruption fragmentation
(sonication)
benefits and drawbacks
Benefits
- accepts wide range of DNA input (nanogram to microgram)
- generates random breaks
Drawbacks
- requires extra specialized instruments and tubes
- often need to sample transfer during the workflow
adapter ligation
enzymatic fragmentation
(fragmentase)
benefits and drawbacks
Benefits
- does not require specialized instruments or tubes
- can be carried out on a regular PCR machine
- don’t need to transfer tubes
Drawbacks
- sensitive to input DNA amount (<1 microgram)
- fragmentation sensitive to reaction time, prone to variations
- sensitive to salt and other potential enzyme inhibitors from sample
- may prefer cutting certain sequences
Adapter ligation
End repair
T4 DNA polymerase creates blunt ends
- 5’ overhang: extends 3’ end of other strand
- 3’ overhang: remove via exonuclease activity
Adapter ligation
Phosphorylation
T4 polynucleotide kinase (PNK) adds phosphate group to 5’ end and removes residual 3’ end phosphate
Adapter Ligation
A-tailing
Taq DNA polymerase adds extra adenine to 3’ end of a double strand DNA molecule without requiring a complementary base
NGS sequencing adapter functions
3
- primer binding site for clonal PCR amplification
- sequencing primer binding
- sample indexing
Adapter ligation
Sequencing adapter
features
- 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
Library prep method #1
Adapter ligation process
- adapter (excess) and end-repaired A-tailed DNA insert are mixed in proper ratio and ligated through T4 DNA ligase.
P5 -> Index 2-> Rd1 SP ->DNA insert -> Rd2 SP -> Index 1 -> P7 - ligation product purified using magnetic beads to remove ligation buffer enzyme and extra adapters
- Low cycle PCR amplification to select for DNA fragments with both adapters
- creates matching double-stranded P5 and P7 ends
- if PCR skipped, may have some DNA inserts with only one or no adaptor ligated - Samples pooled together for loading onto sequencer
adapter ligation
magnetic beads purification
process
-magnetic beads are coated with material that selectively binds DNA based on fragment size
- beads better at binding longer DNA
- by adjusting buffer salt concentration, can tune size of DNA that beads bind to
- beads added to DNA sample and DNA-bead complex formed
- magnetic field is applied, causing the DNA beads complex to migrate to side of reaction tube
- unbound (shorter) DNA fragments remain in supernatant
- supernatant is removed, leaving only DNA beads complex attached to magnet. Removes any unbound DNA fragments, salt and enzymes
- magnetic beads washed with ethanol and released from magnetic field by incubating with elution buffer
- For post ligation purification, salt concentration of beads binding buffer is optimized to bind DNA molecules with adapter and exclude empty adapters
adapter ligation
adapter ligation plate
-each well on adapter plate has adapter with unique index sequence
-can have more than one index per sample
- assign index to samples
- set up ligation with corresponding sample index pairs.
- Once sequencing is completed, reads sorted based on index sequences and assigned to samples according to sample index assignment
Nextera Chemistry
process
- Tn5 transposase conjugated with partial adapter sequences that only include stem part of Y adapter
- Tn5 conjugated with one adapter sequence (partial read 1 or 2) = transposome
- genomic DNA is mixed with transposome in certain ratio.
- transposome cuts double stranded DNA and tags partial adapter to DNA
- Low-cycle PCR with primers matching reads 1 and 2 that have full adapters attached create amplicons with full adapter sequences
transposase
class of enzyme that is capable of binding to the end of a transposon and catalyzing its movement to another part of a genome
transposon
- aka transposable element
- a nucleic acid sequencing DNA that can change its position within a genome (transposition)
Nextera Chemistry
features
- transposase-mediated technique
- fragments and tags DNA in a single step
- size of library insert depends on ratio between genomic DNA and transposome
- sensitive to input DNA amount
- certain sequence preferred by transposase so fragmentation is not completely random
Bead-linked transposome (BLT) kit
- beads covered in transposomes that have Read 1 or Read 2 adaptors
- unfragmented DNA binds to BLT
- transposomes cut DNA and attach Rd1 or Rd2 adapters (tagmentation)
- fragment needs to be ~350bp and have one of each adapter
- low cycle PCR using primers with full adapter sequences
- DNA purified to select for 350bp fragments
- only works well with high-quality long genomic DNA
NGS amplification
Bridging PCR
process
- 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
NGS amplification
Flow cell clusters
- 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
NGS
Read 1 Sequencing
process (9)
- Read 1 sequencing primer anneals to primer binding site
- All four nucleotides, each with different fluorescent label, and polymerase are added to flow cell
- polymerase incorporates first matched nucleotide fluorescently labeled with 3’ end blocked by reversible terminator
- extra nucleotides are washed away and four pictures taken, one for each color, capturing all fluorescent signal from flow cell after first base incorporation
- chemical added to remove fluorescent and blocking groups
- process repeats
- each cluster is at specific location on flow cell and generates one read
- fluorescent signal data analyzed to infer DNA sequence within each cluster
- index primer is annealed to template and reads index 1 (8bp) at end of DNA
NGS
Read 2 Sequencing
process
- molecules denatured to remove newly synthesized DNA strand
- molecules on flow cell undergo bridging PCR
- remove strand attached through oligo complementary to P7
- Read 2 primer anneals to template
- Repeat SBS process
- Index 2 barcode may be sequenced before or after Read 2 bridge amplification
Random (non-pattern) flow cell
features
- have a uniform surface
- Clustering occurs randomly by library molecules binding to flow cell oligos attached to surface.
- critical to precisely quantify library concentration
- PCR enzymes and buffers added after DNA library molecules have annealed to flow cell surface
- residual adapters washed away after hybridization of DNA to flow cell = less index hopping
patterned flow cell
features
- occupied by billions of regularly-spaced nanowells containing flow cell oligos
- library molecules directly deposited into nanowells before clonal amplification
- one molecule per well = 1 cluster per well
- uses exclusion amplification (ExAmp) chemistry
- molecules are deposited into the nanowells randomly as the loading mix flows through entire surface of flow cell
- requires molecules to be amplified immediately after landing in a nanowell to prevent any other molecules getting to same nanowell
patterned flow cell
benefits and drawbacks
Benefits
- cluster generation is more tolerant of wide range of loading concentrations
- minimize risk of over clustering
- no need to map cluster sites
- saves time during sequencing
Drawback
- increase in index hopping events
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
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
pattern flow cell
recombinase polymerase amplification
Exclusion amplification (ExAmp)
- isothermal strand-displacement amplification
- uses recombinase enzyme to help primers invade into double stranded DNA and initiate amplification by DNA polymerase
- avoids needing extension cycles and can generate high amount of amplification products faster than traditional PCR
- more likely to cause index hopping
- more prone to non specific amplification.
NGS detection
4-Channel Chemistry
- bases identified using four different fluorescent dyes for each base, and four images are taken per sequencing cycle
- every sequencing cycle requires four dyes and four images to determine DNA sequence
NGS detection
2-Channel Chemistry
- uses two fluorescent dyes and two images to detect all four bases
- images are taken using red and green filter bands
- Thymines = green
- Cytosines = red
- Adenines = red and green
- Guanines = dark.
- offers faster and cheaper solution for detection, as only two images are required per cycle for detection
NGS detection
1-Channel Chemistry
- Clustering and sequencing occur in nanowells with direct alignment of single clusters over each pixel
- uses 1 dye, 2 chemistry steps, and 2 imaging steps per sequencing cycle.
- adenine = removable label = image 1 only
- cytosine = linker group = image 2 only
- thymine = images 1 and 2
- guanine = dark
- simple and fast detection method supporting lower instrument cost and small instrument footprint
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
prephase
always one base ahead of other strands within the same cluster
- if non-incorporated nucleotide is not completely removed, removal of the blocking group allows addition of extra base
postphase
one base behind other strands in the same cluster
- if removal of blocking group is incomplete, then no nucleotide can be incorporated during next cycle of extension
