11. DNA sequencing methods Flashcards
What are the original DNA sequencing methods?
Original DNA sequencing methods:
- By synthesis (Sanger sequencing)
- Chemical degradation (Maxam-Gilbert method)
Overview of Maxam-Gilbert sequencing
Maxam-Gilbert sequencing:
- based on chemical degradation: base specific cleavage of radioactively labelled DNA 5’ end
- now only used to study DNA-protein interactions
Explain of Sanger sequencing
Sanger sequencing:
- based on synthesis: adding radioactively labelled/dye labelled bases to a primer - base-specific termination of synthesis by ddNTPs (ddATP/ddGTP/ddCTP/ddTTP)
- used for seqeuncing molecularly cloned DNA samples
Explain dye terminator Sanger sequencing
Dye terminator Sanger sequencing:
a fluorescent dye is linked to ddNTPs - 4 diff reactions can be run in same gel lane
What are capillary sequencers?
Automated capillary sequencers use fluorescent dye ddNTPs - run 4 reactions in same capillary lane - can run many samples at once
How can the whole genome, rather than a sequence, be sequenced using Sanger sequencing?
Whole genome sequenced by Sanger:
1) genome clones as large fragment in BAC library
2) overlapping BAC clones identified
3) Each BAC cut into smaller random fragments
4) Fragments randomly cloned into plasmid vectors
5) Plasmids sequenced using universal primer
6) Final sequence assembled from overlaps of sequenced sequences
How are next generation sequencing (NGS) technologies different to original DNA sequencing methods?
Different NGS technologies compared to original methods:
- Sequence DNA directly
- DNA cut into small fragments ~200 bp (ex by sonication)
- DNA fragments immobilised into solid support - DNA molecules physically separated
Explain NGS
Next generation sequencing (NGS):
1) Sample extraction
2) Library preparation: sequence amplification of single DNA + adaptor ligation (for sequence to be able to attact to physical platform) => sufficient ‘polony’/’cluster’ to be detactable
3) Sequencing: parallel sequencing in flow cells where reagents are introduced - reads nucleotides one by one - monitored by detector system (ex fluorescent detector)
4) Alignment and analysis: software used to link read ends - assembled and aligned into a reference genome - further bioinformatic analysis
No product separation by electrophoresis
Overview of Illumina NGS method
Illumina NGS:
- Uses synthesis sequencing principle - DNA pol and fluorescent terminators
- Glass flowcell coated with short oligonucleotides P5 and P7
- ds molecule produced for sequencing
Explain Illumina NGS method
Illumina NGS:
1) Sample preparation: generating DNA library by sonication (DNA fragmented)
2) Cluster generation: ligation to 2 adaptors - ‘bridge amplification’ (cluster amplification) - when enough bridges - denaturation of one strand => high density clusters
3) Sequencing by synthesis: sequencing using dNTPS (dATP, dGTP, dTTP, dCTP) + reversible 3’ - universal primer annealed - DNA pol - sequening of all sites started at once - imaging records fluorescent colour at each position - after imaging dye cleaved => cycle repeated many times for all bases to be sequenced
4) Data analysis: overlapping reads aligned - data anaysed
Describe the physical platform used in Illumina NGS
Illumina NGS uses a glass flowcell - short ss oligonucleotides adaptors (P5, P7) bound to surface or nanowells - dense lawn formed for adaptors (ligated to sequences) to bind to their OH end
The bound oligonucletides will act as primers for DNA polymerysation - bound sequence with adaptors acts as a template strand
Explain the process of sonication
Sonication: using high-frequency sound waves to fragment DNA sequence into smaller pieces
Explain cluster generation in Illumina NGS
When sonicated DNA added:
1) sonicated DNA fragments with ligated adaptors bind to embedded oligonucleotides
2) density of attached DNA adjusted - single DNA molecule at a separated well
3) Initial extension: DNA pol adds dNTPS to make ds DNA from 3’ end - oligonucleotides P5 and P7 act as primers - sonicated DNA as template strand)
4) Denaturation performed - original sonicated DNA washed off - ss copy left
5) Cluster generation: renaturation conditions created - non-bound adaptor bind to another embedded oligonucleotide - bridge formed - DNA pol - another round of DNA synthesis = bridge amplification
=> at each step two strands separated to act as templates for next strand synthesis
Steps 3) -5) repeated x35 times to create an identical sequence cluster in close proximity
Explain sequencing part in Illumina NGS
Illumina sequencing (sequencing of all DNA fragments at once):
- universal sequencing primer annealed to adapter sequences
- DNA pol uses dNTPs with different fluorescent groups: dATP, dGTP, dTTP, dCTP + 3’ reversible block
- incorporation of fluorescent dNTP + temporarily blocks - detector reads fluorescence at each DNA fragment
- the fluore + block removed - new 3’ OH open for next polymerization step - next fluor and block = repeated in cycles until all fragment recorded (leaves the nt but fluore+block removed)
What are the applications fo Illumina NGS?
Explain adapter ligation to sample DNA fragments in Illumina
Adapter ligation: adapters ligated at both ends of DNA fragment - different on each end -> on glass flowcell adaptors bind (base pair) to oligonucelotides P5 and P7 - which act as primers for DNA polymerization (ds to the bound ss sequence)
What is the difference between the primers and oligonucleotides bound to glass flowcells in Illumina?
Primers: bind to sonicated DNA sequences - allow binding to oligos embedded on glass flowcells
Oligonucleotides (P5 and P7): embedded in glass flowcells (the surface) - after binding act as primers for ds DNA synthesis
Why is the density adjusted for only one DNA molecule to bind to glass flowcell well in Illumina?
When sonicated DNA added - density of attached DNA adjusted - single DNA molecule at separated wells => because in the end equal replicates of each sequence needed - ‘bridge amplification’ will amplify each sequence the needed #
How are cluster prepared for sequencing in Illumina?
Cluster preparation for sequencing - before sequencing ds DNA -> ss DNA:
- ddNTPS added to block further binding to oligonucleotides
- at oligo P5 bridge cleaved - one strand removed by denaturation
- a sequencing primer bound
What is used to separate different DNA clusters on glass flowcell in Illumina?
Distance - identical sequence clusters are separated by distance on the glass flowcell
Explain the structure of the glass flowcell in Illumina
What techniques can be used to study chromatin structure, transcriptional regulatory regions, TF binding?
Study chromatin structure, transcriptional regulatory regions, TF binding:
- DNase I hypersensitivity mapping (DH mapping at DHS) - footprinting-like for genomw wide scale - looks at loose chromatin regions not which bound
- Chromatin immunoprecipitation (ChIP)
What is the basis for DNase I hypersensitivity of chromatin?
DNase I hypersensitivity of chromatin:
- more accessible chromatin regions are more sensitive to DNase I cleavage - DNase I hypersensitivity sites (DHS) => indicates open active chromatin
What is studied using DNase I hypersensitivity of chromatin?
DNase I hypersensitivity of chromatin used to study gene regulation (enhancers, promoters) in gene expression
Ex used in developmental studies - which gene active at what stage and in which cells
What is the sequence of DNase I hypersensitivity of chromatin method?
DNase I hypersensitivity of chromatin method:
1) isolate cell nuclei
2) add DNase I
3) stop reaction with SDS + protease (denatures DNase I enzyme)
4) purify DNase I cut genomic DNA
5) cut with different restriction enzymes
6) Southern blot with different probes
What is the basis for chromatin immunoprecipitation (CHIP)?
Chromatin immunoprecipitation (CHIP) uses selective DNA enrichment of fragments associated with particular protein - add Ab for the bound proteins => indicates where TF bind, chromatin modifications
What is the sequence of CHIP method?
Chromatin immunoprecipitation (CHIP) method sequence:
1) Cross-linking: protein-DNA covalent bonds
2) Cell lysis: release nuclear contents
3) Fragmentation: chromatin fragmented (to be accessible for Ab)
4) Immunoprecipitation: Ab for bound protein added - chromatin-Ab complex = immunoprecipitate
5) Washing and elution: protein-DNA crosslink broken
6) DNA purification: DNA purified from protein and Ab mixture
7) DNA analysis: PCR / sequencing to find out what sequence was bound by the specific protein
What is the appropriate sequencing method to analyse DNA from DHS and CHIP?
Total DNA from DHS and CHIP analysed by NGS - can read many sequences => CHIP-seq, DNase-seq
- Used for genome-wide chromatin studies
- Short sequences (25-35 bp) needed to map location to reference genome
- Softwares count read numbers - generate peaks to analyse distribution through genome
Explain DNase-seq
DNase-seq - after open chromatin regions identified using DNase I - sequenced:
1) digested closed DNA purified
2) library preparation - fragment end repair, adapter ligation, PCR amplification
3) Sequencing: NGS (ex Illumina)
4) Analysis: sequence reads aligned to a reference genome - location identification of abundant reads - see which missing => active gene identification + their spatial distribution
Explain CHIP-seq
CHIP-seq - after protein bound DNA fragments identified using Abs - sequenced:
1) DNA purified from Abs and proteins
2) Library preparation: DNA end repairing, adaptor ligation, PCR amplification
3) Sequencing: NGS (ex Illumina)
4) Analysis: sequence reads aligned to a reference genome - location identification of the genes + their spatial distribution
What is a reference genome in sequencing?
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