Next Generation Sequencing Flashcards
Principal of Sanger Sequencing (Dideoxy sequencing)
- First DNA sequencing method and used since 1977
- Like most sequencing methods, it is template based
- start with a single strand of DNA, which is produced by using a single primer
Set up:
four “sequencing reactions” wich contains:
–> DNA template
–> DNA Polymerase
–> Primers
–> Nucleotides (dNTPS)
–> A lown concentration of one fluorescently-labelled dideoxy nucleotide ddNTPS (A,T,G or C)
–> 4 Tubes with 4 diffrent ddNTPS
Tube 1: ddATP
Tube 2: ddTTP
Tube 3: ddGTP
Tube 4: ddCTP
Method:
1. DNA is denatured
2. Primer bind to DNA region of interest
3. In present of 4 nucleotides the Polymerase will extend the primer by adding on the complementary nucleotide from template DNA strand
4. Reaction stops when the di-deoxy nucleotides is added to DNA strand
different chains will be different lengths
- they can be separated by gel electrophoresis (denaturating polyacrylamide gel electrophoresis)
- First: Separate your four reactions on four lanes of a large gel, DNA polymerase + 4 dNTPs (ddC, ddT, ddA, ddG)
- Now: DNA is labeled with 4 different fluorophores. Meaning you could run 1 sample per lane. Detection of fluorescence
What are ddNTPS?
Dideoxy nucleotide is missing 1 oxygen atom at Ribonucleotide.
Without the 3´OH group the reaction stops because there wont be addition of new nucleotides in the polynucleotide chain
Principal of Ilumina Sequencing?
- Sequenicng by synthesis
Material:
* Flow cell with fixed primers
* DNA of interest
* Additional Adapters
* Labeled Nucleotides
* Polymerase
Sample prep:
* extract DNA of interest
* Fragementation of DNA
* Denaturation of DNA
* Ligation of Adapters to Fragements
Cluster Generation:
* Oligonucleotides that are complementary to adapters are fixed on Flow cell
* DNA fragments bind to oligonucelotides on Flowcell
* Polymerase creates complementary DNA strand (starting from fixed oligonucleotide) building dsDNA
* dsDNA is denaturated
* original (not fixed) DNA is washed away
* Bridge amplification leds to colonie amplification of strands
Bridge amplification = DNA strain folds over, Adapter hybridzes to oligonucleotide that is fixed on flowcell. Polymerase creates dsDNA. Denaturation.
–> Foward Strand
–> Reverse Strand (both fixed on Flow cell)
This process is repeated several times. Leads to Polonies of DNA strand
Polonies= polymerase generated colonies, that can be simultaneously
sequenced
* At the End we have a lot of Reverse strand and Foward strand
* Reverse strands are cleaved and whased away
Sequencing:
1. dNTPs (Four fluorescent labeled nucleotides allow detection via pictures) and primers are added.
2. First sequencing cycle:
dNTPS are added to DNA strands.
–> only the latest added dNTP is fluorescent. Image taken after each cylce.
4. Sequence is derived from images.
5. Reads are up to ~100 bases.
6. After reading all soward strands
7. A new Bridge amplification takes place
8. This time all Foward strands are washed away
9. Second sequencing cylce …
Quantification
* All identical strands are read simultanously
* Billion of reads are generated
* Foward and Reverse reads are paired = contigous sequences
* contigous sequences are alignet back to refrence genome for identification
* Programs like R help to identify sequences
Potential limitations of Illumina sequencing technologies
Disadvantages:
* Only short sequence reads
* Must be assembled computatationally
* Difficult for repetitive sequences (not suitable for competitive parts in genome)
* gaps called contigs
* quality declines with higher read length
Advantages: high throughput and high sequencing quality, relatively cheap
Principle of PacBio sequencing
Pacific Biosystem:
* Real time sequencing
SMRTbell Preparation:
* DNA gets fragmented in nebulizer, ends are polished.
* All double stranded DNA fragments receive bell-like adapter on both ends.
* DNA polymerase starts extension at primer goes along the DNA
–> opens the double strand
-> one side gets replicated other side now single strand
–> goes around the molecule
–> pushes away primer
–> can move around circular template several times
–> multiple copies of the same template.
SMRT-cell: with Zero Mode- Waveguides
* One active polymerase is immobilized at the bottom of the reaction chamber.
* Each nucleotide labeled with a differend fluorescent colour.
* Only the bottom of the well is lluminated
–> only nucleotides near bottom are fluorescent.
* Nucleotides are labeled fluorescently through phosphate groups
* label removed once nucleotide is attached to growing DNA strand.
What are the major innovations in PacBio sequencing compared to other/previous
sequencing technologies?
Advantages:
* Real-time
* unique SMRTbell Library Preparation
* Squencing perfored on SMRTcell (small wells on a chip conatina thousand of zero-mode waveguides. Each ZMW can accomodate a single DNA template for sequencing)
* Long Read Lengths
* High Accuracy with circular censensus sequencing
* No washing: cheap on reagents
* Strictly, single molecule reaction monitoring
* you can also sequence nucleotides with modificvations e.g: 5-methylcytosine
(In sanger sequence you cant tell the diffrence between cytosin and 5-methylcytosin –> both is cytosin)
Disadvantages:
* Initially high error rate
Detection of DNA modifications in genomic DNA (epigenetics)
- Sequencing of 5-methylcytosin (5mC) is possible
Method: - all cytosin residues are converted to uracil
- 5mC is not converted to uracil
- Sequencing for A,T,G,C and U
- PacBio can detect modified bases by analyzing variation in the time between base incorporations in the read strand
Principle of Ion Torrent sequencing
- Measure something that is normally not determined
- Each time a nucleotide is incorporated a hydrogen ion is released.
- Each well hold a different DNA template.
- Each nucleotide is added separately.
- Is a nucleotide is added and incorporated a hydrogen ion is released.
- The machine measured the pH.
- If two are incorporated the spike is higher.
- All nucleotides of a type are washed away before the addition of the next one
Advantage:
* Fast and cheap
Disadvantage:
* high error rate
Principle of nanopore sequencing
- Based on threading a single strand of DNA through a microscopic hole in a membrane
- no PCR amplification needed
- Creating an electric field across the membrane causes the DNA to pass through
- A DNA fragment moves through a protein pore
- Measuring the electrical properties of the hole (capacitance pA meter), should tell you which base is passing through it
- Speed: 200-500 bases per second per pore
Advantage:
No need for amplification,
can be used in field experiments
real-time results
Disadvantages:
High error-rate, biased
Principle of chromosome confirmation capture sequencing; for what is it used?
Method to analyse spatial organisation (3D) of chromatin and measure interactions between genomic loci that are neighboring in 3D space.
–> stduying linkage&proximity
- Chromatin is packed into 3D structures that retain a relationship between genomic and physical distance
Method:
* DNA in sample is crosslinked using formaldehyd
* cross linking trap sequenc interactions across the entire genome + between diffrent cells
* cross linked DNA is fragmented with endonucleases
* framented loci are then biotinylated and ligated
–> creating chimeric junctions between adjacent sequences = proximity ligation
* The Joint fragments are then sequenced
* biotnylated junctions are purified and subjected to pair end sequences
Analyzing:
* The more often 2 sequences are joined together, the closer these 2 sequences are in genomic space
* The proximity ligation reads are then mapped onto draft assembly
* used to assign contigs to chromosome and to learn which part of the chromosmes sits together
Principle of ATAC-seq; for what is it used?
A method for determining chromatin accessibility across the genome.
–> Chromosome accesability varies depending the genomic region but also between cells
unwrapped and free DNA = transcriptionally activ
ATAC-Seq=Assay for Transposase Accessible Chromatin
method
* Genomic DNA is isolated and then incubated with transposase T5
* T5 binds to accessable DNA Regions and adapters are added
(T5 = an enzyme which cleaves and adds adapters to the nucleosome-free regions of the DNA (tagmentation)).
* The DNA fragments (that where accessable fro T5) are sequenced (NGS)
–> Shows which region of chromosome is accessable
–> helps to map nucleosomes and open chromatin regions
