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