Genomics : DNA Sequencing Flashcards
Why sequence the whole genome?
- Provides complete genetic information of an organism.
- Detects mutations, SNPs, and structural variations across the genome.
- Helps identify genetic causes of diseases.
- Enables tailored treatments based on genetic profiles.
- Reveals genetic relationships and evolutionary history.
What makes up a nucleotide?
- Phosphate group: Linked to the sugar molecule.
- Sugar: Deoxyribose in DNA, ribose in RNA.
- Nitrogenous base: One of four types:
- Purines: Adenine (A),
Guanine (G)
- Pyrimidines: Cytosine
(C), Thymine (T) in DNA;
Uracil (U) in RNA.
What bond is formed when 2 nucleotides join together?
- Phosphodiester bond:
- Forms between the phosphate group of one nucleotide and the sugar of another.
- Links the 3’ carbon of one sugar to the 5’ carbon of the next sugar.
What is the principle of Sanger Sequencing?
Sanger sequencing works by using special building blocks of DNA, called dideoxynucleotides (ddNTPs), which act as “stop signals” during DNA synthesis
How is the Sanger Library prepped?
- DNA Extraction: Isolate the DNA of interest from cells or tissue.
- Fragmentation: The DNA is fragmented into smaller, manageable pieces using enzymes or mechanical methods.
- Adaptor Ligation (if needed): Short, known sequences (adaptors) may be ligated to the DNA ends for primer binding.
- Cloning into Vectors: DNA fragments are inserted into plasmid vectors for amplification in bacterial cells.
- Transformation: The recombinant plasmids are introduced into competent bacteria.
- Amplification and Isolation: Bacterial colonies are grown, and plasmid DNA is extracted to isolate the cloned DNA fragments.
- Template Preparation: Purified DNA is used as the template for sequencing.
What is mechanical DNA shearing?
Mechanical DNA shearing is a method used to fragment DNA into smaller pieces through physical forces. It creates random breaks in DNA, making it useful for preparing DNA libraries for sequencing.
What are the common methods of mechanical shearing?
- Sonication
- Ultrasonic waves are used to create vibrations that shear DNA.
- The length of fragments depends on the duration and intensity of sonication.
- G-Tube
- forcing DNA through a narrow constriction (the G-tube), creating consistent shear forces that fragment the DNA into specific, predictable sizes. - Hydrodynamic Shearing:
DNA is passed through a constricted tube, and the sudden pressure change causes shearing.
What is enzymatic DNA shearing?
Enzymatic DNA shearing is a method of DNA fragmentation that uses enzymes like DNase I or endonucleases to cleave DNA into smaller fragments.
The fragment size can be controlled by adjusting reaction time and enzyme concentration.
What is the importance of Sanger Sequencing?
Gold standard for accuracy: provides highly accurate, low error rate DNA sequences. Used to validate results from high-throughput NGS
Small-Scale Applications: suitable for sequencing small DNA fragments, used in clinical diagnostics, gene cloning and identifying mutations
What is next-generation sequencing?
an advanced sequencing technology for determining the sequence of DNA or RNA to study genetic variation associated with diseases or other biological phenomena.
It uses the concept of massive parallel processing.
What is the 454 Library Prep?
454 Library Preparation is a method used in 454 sequencing (a type of Next-Generation Sequencing) to prepare DNA fragments for sequencing by attaching adapters, amplifying the DNA, and preparing it for pyrosequencing.
Explain the key steps in 454 Library Prep.
- DNA Fragmentation:
- DNA is sheared into
fragments of 300–800 base
pairs (bp).
- Ends are polished by
removing unpaired bases to
create blunt ends. - Adapter Ligation:
- Adapters (named A and B)
are attached to both ends of
the fragments.
- DNA is made single-
stranded at this stage. - Bead Attachment:
- One adapter contains biotin,
which binds to streptavidin-
coated beads.
- The ratio of beads to DNA is
controlled to ensure that
each bead gets only one
DNA molecule attached. - Emulsion PCR:
- Oil is added to the beads,
creating an emulsion where
each aqueous droplet serves
as a micro-reactor.
- PCR is performed within
these droplets, amplifying
the DNA.
- Each bead ends up coated
with millions of identical
copies of the original DNA
fragment. - Preparation for Sequencing:
- The DNA-coated beads are used in 454 sequencing for pyrosequencing reactions.
Explain the brief chemistry on 454 sequencing.
- Nucleotide Incorporation:
When a base (dNTP) is incorporated by DNA polymerase, pyrophosphate (PPi) is released.
- Conversion to ATP:
PPi reacts with adenosine 5’-phosphosulfate (APS) in the presence of the enzyme sulfurylase, generating ATP.
- Light Signal Production:
Luciferase uses ATP to convert luciferin into oxyluciferin, releasing light as a byproduct.
The amount of light is proportional to the number of bases added (e.g., a homopolymer of “AAA” will produce a stronger signal).
- Degradation of Unused Reagents:
The enzyme apyrase degrades excess ATP and unused dNTPs before the next nucleotide cycle begins.
- Flowgram Analysis:
The light signals are recorded as peaks on a flowgram, with each peak corresponding to the nucleotide added (A, T, G, or C).
Flowgrams are used to reconstruct the DNA sequence.
454: why is it not commonly used anymore?
454 sequencing has largely been replaced by more advanced next-generation sequencing (NGS) technologies due to several limitations:
cost, error rate, and scalability, combined with the emergence of better alternatives,
Explain the steps in enzymatic DNA shearing.
- Preparation and Input DNA:
Start with a minimal DNA input; often as low as 1 nanogram is sufficient.
This method works for small genomes, amplicons, and plasmids.
- Enzymatic Reaction:
DNA is incubated with an enzyme cocktail optimized to cleave at specific points.
The reaction time, temperature, and enzyme concentration are controlled to achieve desired fragment sizes.
- Normalisation of DNA:
Innovative sample normalization eliminates the need for manual library quantification.
This step ensures consistent DNA input across samples.
- Adapter Ligation (if applicable):
Once sheared, DNA fragments are ligated to adapters that are required for downstream sequencing.
- Library Preparation and Amplification:
The prepared DNA fragments are amplified using PCR to generate a sequencing-ready library.
- Sequencing:
Prepared DNA is loaded into a sequencing platform (e.g., MiSeq) for analysis.
Rapid Prep: The entire workflow from DNA to data can be completed in less than 8 hours.
What is sample pooling?
Sample pooling is a method in DNA sequencing where multiple DNA samples are combined into a single sequencing run. Unique identifiers (indices) are added to each sample to differentiate them during analysis.
What is indexing/barcoding?
Indexing or barcoding is a technique in DNA sequencing where unique DNA sequences (indices) are added to multiple samples, allowing them to be sequenced together (multiplexed) and computationally separated (demultiplexed) later.
What are the advantages and disadvantages of indexing/barcoding?
Advantages:
Reduces Costs: Multiple samples can be processed in a single run, saving on reagents and sequencing costs.
Faster Turnaround Time: Speeds up processing as multiple samples are sequenced simultaneously.
Disadvantages:
Reduced Read Number per Sample: Since all samples share the same sequencing capacity, the number of reads per sample is lower.
Normalization Required: Ensures equal representation of each sample to minimize variation in read numbers.
Illumina - What is cluster generation?
Cluster generation is a process in Illumina sequencing where individual DNA fragments are amplified in a flow cell to create clusters of clonal DNA molecules.
These clusters generate enough fluorescent signals during sequencing to be detected accurately.
Steps in Cluster Gen
- DNA Binding to Flow Cell:
DNA fragments, ligated with P5 and P7 adapters, bind to complementary oligonucleotides on the flow cell surface.
- Bridge Amplification:
The DNA fragment bends and hybridises to nearby oligos, creating a “bridge.”
PCR amplifies the DNA, forming double-stranded molecules.
- Denaturation and Clonal Amplification:
Double-stranded molecules are denatured into single strands.
The process repeats, creating clusters of identical DNA molecules.
- Purpose:
Amplify enough DNA to ensure the fluorescent signal from incorporated nucleotides during sequencing is strong enough for detection.
What is the purpose of P5 and P7 adapters in Illumina sequencing?
P5 and P7 adapters allow DNA fragments to bind to the flow cell and serve as primers for PCR amplification during cluster generation.
Why is bridge amplification important?
Bridge amplification creates clusters of clonal DNA, ensuring that each cluster generates sufficient signal for accurate sequencing.
What is the primary goal of cluster generation?
To amplify individual DNA fragments into clusters, producing enough signal for sequencing by synthesis (SBS).
How does Illumina ensure each cluster represents a single DNA fragment?
By using precise ratios of DNA and oligonucleotide sites on the flow cell, each DNA molecule is amplified into a distinct cluster.
Illumina - What is the chemistry behind this?
Illumina sequencing uses sequencing-by-synthesis (SBS), which involves the incorporation of fluorescently labeled nucleotides to build a DNA strand one base at a time.
What are the key steps in Illumina chemistry?
- Dye-Termination Similar to Sanger Sequencing:
DNA synthesis starts with a primer.
Each added nucleotide is fluorescently labeled, emitting a signal when incorporated.
- Blocking of the 3’-OH Group:
Each nucleotide has a reversible terminator that blocks the 3’-OH, preventing further base additions.
After imaging, the fluorescent dye and blocking group are removed, allowing the next nucleotide to bind.
- Fluorescent Signal Detection:
A camera captures the signal emitted by the fluorescent dye, identifying which nucleotide (A, T, G, or C) was incorporated.
Unlike pyrosequencing, Illumina avoids issues with homopolymer stretches because it processes one base at a time.
- Cycle Repetition:
The process is repeated 50–300 times, extending the DNA strand and generating sequence reads.
How does Illumina differ from pyrosequencing?
Illumina uses reversible terminators and avoids issues with homopolymer stretches, whereas pyrosequencing relies on pyrophosphate detection, which can misread homopolymers.
What is the role of reversible terminators in Illumina sequencing?
They block further nucleotide incorporation until the dye and blocking group are removed, ensuring one base is added at a time.
Why is fluorescence used in Illumina sequencing?
Fluorescent signals allow precise detection of which nucleotide is added during each cycle.
What is the significance of paired-end indexed sequencing
Paired-end indexed sequencing is a method in which both ends of a DNA fragment are sequenced, providing more comprehensive information about the DNA sequence and its context. It is particularly useful for discovering genome variation and improving sequencing accuracy.
