Genome Sequencing Midterm 2 Notes
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Questions and Answers

What occurs when Sgrna binds to DNA?

  • It promotes DNA replication.
  • It prevents the DNA from being cleaved.
  • It initiates transcription of the DNA.
  • It displaces the duplex and creates a single-stranded DNA region. (correct)

What is the role of Cas9 after Sgrna binds to the DNA?

  • It facilitates the binding of additional proteins.
  • It inhibits DNA repair mechanisms.
  • It seals the DNA after cleavage.
  • It cleaves DNA at the PAM site. (correct)

Which method is NOT mentioned for delivering Cas9 and Sgrna into the nucleus?

  • Plasmids transient method in mammalian cells.
  • Direct injection of Cas9 protein into the nucleus. (correct)
  • Viral particles introducing DNA coding for Cas9 and RNA.
  • In vitro purification of Cas9 and RNA for introduction.

How does the precision of DNA-RNA interactions compare to DNA-protein interactions like zinc fingers and TALENs?

<p>DNA-RNA interactions are generally more precise. (A)</p> Signup and view all the answers

What is a key advantage of using Sgrna and Cas9 over zinc fingers or TALENs?

<p>It requires fewer nucleotides to change. (A)</p> Signup and view all the answers

What is the purpose of the complementary binding in the sequencing process?

<p>To create a phosphodiester bond (A)</p> Signup and view all the answers

What is the ideal sequence read length mentioned?

<p>150 bp (C)</p> Signup and view all the answers

What role does the cleavable dye play in the sequencing process?

<p>It is attached to the phosphate group (A)</p> Signup and view all the answers

What distinguishes the Ion Torrent sequencing method from others?

<p>It is the first platform to sequence DNA without fluorescence (D)</p> Signup and view all the answers

Why is a free 3' OH group necessary in the sequencing process?

<p>To facilitate phosphodiester bond formation (C)</p> Signup and view all the answers

How does the Ion Torrent method detect nucleotide incorporation?

<p>By detecting changes in voltage (D)</p> Signup and view all the answers

What is required to generate the reverse complement of the DNA strands in the sequencing process?

<p>Denaturation and bridging of the strands (B)</p> Signup and view all the answers

What is the change in pH that occurs when a nucleotide is added in Ion Torrent sequencing?

<p>0.02 unit change (A)</p> Signup and view all the answers

What is a significant advantage of long reads in genomic sequencing?

<p>They allow for quicker genome assembly. (A)</p> Signup and view all the answers

How does nanopore sequencing primarily function?

<p>By using a membrane with pores and motor proteins. (D)</p> Signup and view all the answers

What does the term 'squiggle' refer to in nanopore sequencing?

<p>The modulation of electrical current based on base structure. (D)</p> Signup and view all the answers

What percentage of disease-causing variants arise from exons in whole exome sequencing?

<p>85% (D)</p> Signup and view all the answers

Which of the following modifications can nanopore sequencing detect?

<p>DNA methylation on cytosines. (D)</p> Signup and view all the answers

What is one limitation of using reference genomes for sequencing data?

<p>They may not capture variability present in populations. (D)</p> Signup and view all the answers

Why is next-generation sequencing (NGS) important even after the human genome has been sequenced?

<p>It helps in identifying genetic causes of diseases. (C)</p> Signup and view all the answers

What is the role of DNA helicase in nanopore sequencing?

<p>To unwind DNA and aid in feeding it through nanopores. (D)</p> Signup and view all the answers

What is the primary function of the polymerase during the detection step?

<p>To add complimentary nucleotides (A)</p> Signup and view all the answers

Which of the following is NOT a step in the GWAS process?

<p>Identifying genome-wide rare mutations (C)</p> Signup and view all the answers

What role does the primer play in DNA sequencing?

<p>It provides a 3’OH group for DNA polymerase to initiate replication. (B)</p> Signup and view all the answers

In the context of genome-wide association studies, what does a Manhattan Plot illustrate?

<p>A view of all SNPs with their statistical significance (D)</p> Signup and view all the answers

What is a key characteristic of the 4th generation sequencing method?

<p>It reads nucleotide sequences directly from the DNA molecule. (D)</p> Signup and view all the answers

What is the role of bio banks in genetic research?

<p>Distributing and accepting biospecimens (D)</p> Signup and view all the answers

What is the purpose of having multiple stages in the GWAS approach?

<p>To enhance detection of significant SNPs (D)</p> Signup and view all the answers

How does the use of multiple reads contribute to the sequencing process?

<p>It allows the creation of a consensus sequence to improve accuracy. (C)</p> Signup and view all the answers

What happens to the fluorescence signal during the DNA sequencing process?

<p>It decreases after the phosphodiester bond is formed. (D)</p> Signup and view all the answers

Which of the following describes pharmacogenomics?

<p>Study of genetic influences on drug metabolism (A)</p> Signup and view all the answers

What could potentially occur if a GWAS is too stringent with its p-value criteria in the first stage?

<p>Missed potential significant SNPs (A)</p> Signup and view all the answers

What is the role of bioinformatic tools in the sequencing process?

<p>They separate, trim, and remove adaptor sequences from read data. (A)</p> Signup and view all the answers

Why is real-time sequencing considered advantageous?

<p>It allows for continuous measurement without additional steps. (C)</p> Signup and view all the answers

What is represented by the term PAR in genetic studies?

<p>Proportion of incidence attributable to exposure (D)</p> Signup and view all the answers

What modification is present on nucleotides used in this sequencer?

<p>They are fluorescently labeled on their phosphates. (B)</p> Signup and view all the answers

What does the bottom of the wells in DNA sequencing contain that aids the detection process?

<p>Transparent materials to allow fluorescence measurement. (A)</p> Signup and view all the answers

What is the main difference between Sanger sequencing and Illumina sequencing regarding the nucleotides used?

<p>Sanger uses fluorescent nucleotides as chain terminators, whereas Illumina uses reversible terminators. (A)</p> Signup and view all the answers

Why is sequencing performed from top to bottom in the context of strand orientation?

<p>The 3' end of the template strand is at the top, allowing synthesis at the top. (B)</p> Signup and view all the answers

What role do the microfluidic channels play in the Illumina sequencing process?

<p>They help in introducing all DNTPs at once. (D)</p> Signup and view all the answers

In Illumina sequencing, what happens after imaging the fluorescent signal?

<p>The 3' OH of the modified nucleotide is unblocked and the fluorophore is removed. (A)</p> Signup and view all the answers

What is a key feature of the fluorescent nucleotides used in Sanger sequencing?

<p>They terminate the DNA chain due to the absence of a 3’ OH. (A)</p> Signup and view all the answers

What is the purpose of washing away unincorporated nucleotides in the Illumina sequencing process?

<p>To ensure only one nucleotide is added in each cycle. (C)</p> Signup and view all the answers

Which of the following statements correctly describes the role of DNA ligase in sequencing by ligation?

<p>It ligates the DNA fragments to create longer sequences. (C)</p> Signup and view all the answers

Which component in Illumina sequencing aids in signaling the presence of incorporated nucleotides?

<p>Reporter dyes. (B)</p> Signup and view all the answers

Flashcards

Sequencing by Synthesis

A DNA sequencing method where DNA is synthesized and sequenced at the same time using fluorescently labeled nucleotides.

Illumina Sequencing

A sequencing method using reversible terminators, blocked 3'OH, a reporter dye, and a cleavage site.

Reversible Terminators

Modified nucleotides used in Illumina sequencing that block the 3'OH for chain termination but can be unblocked for continuation.

Fluorescence-based Method

Sequencing method depending on fluorescent signal detection of added nucleotides.

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Sequence from top down

To determine the sequence order of DNA segments by synthesizing new DNA strands on the surface starting from the top of the template.

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Complementary Strands

Two DNA strands with base-pair matches that are antiparallel.

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Microfluidic Channels

Channels in Illumina sequencing that allow controlled introduction of dNTPs.

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dNTPs

Deoxynucleoside triphosphates, the building blocks of DNA.

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Paired-end sequencing

Sequencing both ends of a DNA molecule (sense and anti-sense).

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Oligo lawn

A surface containing both types of oligonucleotides needed for sequencing.

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Ion Torrent sequencing

A type of DNA sequencing that detects changes in pH caused by nucleotide addition and analyzes single nucleotide editions

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Sequencing by synthesis

A DNA sequencing method where nucleotides are added one at a time to the growing DNA chain.

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150bp ideal sequence read length

Optimal DNA fragment size for efficient paired-end assembly.

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Phosphodiester bond

A chemical bond formed between nucleotides during DNA synthesis.

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Change in pH

A change happens in pH and the voltage.This voltage change is measured.

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Nucleotide addition

Process of adding a nucleotide to the growing DNA chain in sequencing by synthesis.

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Long Reads Sequencing

A DNA sequencing method that generates longer DNA sequences, improving genome assembly.

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Nanopore Sequencing

A method that sequences DNA by measuring changes in electrical current as DNA passes through a tiny pore.

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DNA Methylation

A process where a methyl group is added to a DNA molecule, often influencing gene expression.

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Whole Exome Sequencing

A method of sequencing that focuses on the exons (coding regions) of a genome.

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Exome

The portion of a genome that contains exons, the coding regions of genes.

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Genome Variability

Differences in DNA sequences among individuals of a species.

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NGS in Paleontology

Use of next-generation sequencing to study evolutionary relationships, ancestry, and hereditary patterns in ancient DNA.

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Reference Genomes

A representative set of DNA sequences used to compare and analyze other genomes. Representative DNA sequences created from sequencing.

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SMRT Sequencing

A fourth-generation DNA sequencing technology that directly reads the nucleotide sequence from the DNA molecule without copying.

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Raw Read Length

The length of information acquired after initial sequencing from a single molecule in SMRT seq.

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Circular Consensus Sequencing

A bioinformatics procedure to create a consensus sequence combining multiple raw reads for increased accuracy, adjusting for potential errors during sequencing.

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Real-time Sequencing

SMRT Sequencing, which continuously measures fluorescence as nucleotides are added, avoids intervening steps and needs no separate signal cleavage.

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DNA Polymerase Immobilization

DNA polymerase is fixed to the sequencing well to facilitate real-time detection of nucleotide inclusion during sequencing.

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Fluorescent Labeling

Modified nucleotides in SMRT sequencing have fluorescent labels that are detected when incorporated by the DNA polymerase.

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Template Binding

The template strand of DNA binds to the immobilized DNA polymerase starting the sequencing process.

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Error Rate (Sequencing)

Although SMRT sequencing generates long reads, the accuracy of a single read is less than other sequencing methods.

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GWAS

Genome-Wide Association Study; a method to identify genetic variations associated with diseases or traits.

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sgRNA binding

sgRNA binds to DNA upstream of the PAM sequence, displacing the DNA duplex to create an sdna region, guiding Cas9

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BeadChip

A microarray device used in GWAS to genotype many SNPs simultaneously.

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Cas9 cleavage

Cas9 cleaves DNA 3 nucleotides upstream of the PAM sequence, always in the 5' to 3' direction

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SNP

Single Nucleotide Polymorphism; a variation in a single DNA nucleotide.

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Cas9 delivery

Cas9 and sgRNA can be delivered to cells using plasmids, viral vectors, or purified pre-assembled complexes

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Cas9 vs. ZFN/TALEN

Cas9-sgRNA systems are more precise and easier to design than zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) for gene editing

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Manhattan Plot

A graphical representation in GWAS visualizing SNP associations.

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target region

Specific region of DNA that is meant to be targeted by the guide RNA (gRNA)

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Polymerase Extension

DNA polymerase adds nucleotides to an extending probe (oligonucleotide) that is linked to the target.

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Fluorescent Nucleotides

Nucleotides labeled with fluorescent markers used in sequencing to detect nucleotide incorporation.

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Multi-stage GWAS Approach

A stepwise approach to GWAS, starting with a large number of SNPs, then refining with more stringent criteria to confirm findings.

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Precision Medicine

Using genetic information to tailor treatments to a patient's specific genetic makeup.

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Study Notes

Midterm 2 Notes (October 2nd)

  • Cheaper genome sequencing uses BAC clone libraries and shotgun sequencing methods instead of hierarchical steps, increasing efficiency by using parallel sequencing and shrinking reagent sizes.

  • Next-generation sequencing (NGS) methods use integration, parallelization, and miniaturization for efficiency. These methods sequence millions or billions of sequences simultaneously, requiring only one sequencer.

Workflow

  • Prepare Library
    • Starts with genomic DNA extraction (100-500ng).
    • Fragments DNA into random pieces (200-400bp) for assembly.
    • Ligates adapters to fragments. There are specific adapter sequences (P5/P7) for binding to the sequencing surface. These adapters are short, double-stranded DNA fragments.
    • Size selection is important for instrument capacity.
    • Amplification is optional for higher sequencing material
    • Library is ready for sequencing.

Sequencing Library

  • Hierarchical steps are skipped in NGS
  • Clones aren't used
  • All DNA is fragmented, and adapters are added
  • DNA is attached to a solid surface
  • The patterned flow cell allows for separate DNA templates.
  • Illumina sequencing synchronously sequences clusters at a set rate.
  • Each flow cell can sequence 400 million templates (2 billion in sequencing overall). Templates are attached to a surface and a DNA polymerase builds complementary strands on the template. DNA is then denatured, and a new strand is built.

Other Information

  • Size selection is important for preparing DNA for sequencing
  • Amplification is optional to increase the amount of DNA available
  • Sequencing is done using a fluorescence-based method; different nucleotides have different colors
  • Illumina sequencing: Fluorescent nucleotides are used; they're blocked in 3' OH to ensure only one nucleotide is added at a time.
  • Fluorescent signal is read to determine sequence

Stage 2 Summary

  • Amplify or size select DNA
  • Use primers to recognize specific DNA
  • Remove material without the primers attached
  • Begin sequencing stage, with additional steps
  • Determining sequence length
  • Detecting differences

Real-Time Sequencing

  • Process is continuous, no intervention needed between signaling stages
  • Faster and longer reads than previous methods

Nanopore Sequencing

  • Membrane with pores and a motor protein (DNA helicase) aids in feeding DNA through the pore
  • Current is modulated in relation to the base sequence
  • Machine learning algorithms decode the modulated current

Other Important Notes

  • DNA methylation
  • Variants: A subset of DNA sequence variations that occur naturally. Some are damaging, but most have a neutral effect on physiology or disease.
  • SNPs: (Single nucleotide polymorphisms) are the most abundant type of variation in humans. They are a substitution of one nucleotide for another.
  • CNVs: (Copy number variations) are a change in the number of copies of a DNA segment.
  • Population-level Sequencing (1000 Genomes Project): A project to catalog human genetic variation

3rd Generation Sequencing

  • Single molecule real-time sequencing (SMRT) uses long reads for entire genome sequencing
  • Ligation of adaptors creates a circular configuration for DNA.
  • DNA polymerase extension is used to synthesize the new DNA strand.
  • DNA polymerase is used on a single strand of DNA; the synthesized molecule is kept and the opposite strand is thrown away when it's no longer needed.

Genome-Wide Association Studies (GWAS)

  • Used to catalog human genetic variation and link certain genomic variants to specific diseases.
  • Large scale genome-wide scanning for polymorphic markers to identify variants correlated with diseases.
  • Large-scale sequence analysis to scan variation associated with specific diseases.
  • Focuses on protein-coding sequences.

October 28th Notes

  • Looking at structural variants and how they relate to chromosomes.
  • MCNVs and duplications are associated with regions possessing repetitive elements, especially at telomeres and centromeres.
  • Summary of findings in the paper and why genomic variants are important to study.

October 30th Notes

  • Genome-wide association studies (GWAS) compare genome sequences to identify genetic variants associated with similar phenotypes.
  • The focus is identifying variants correlated with diseases.
  • Uses tag SNPs, which represent a haplotype and are often in linkage disequilibrium, thus are correlated to each other.

Other Notes

  • Base Editing
    • Efficiently alters DNA sequences.
    • Utilizes a fusion protein composed of a Cas9 enzyme and a deaminase. This targeted process has a lower likelihood of off-target effects.
  • Homology-directed repair (HDR)
    • Uses a DNA template to change the DNA sequence. A homologous sequence is added to the genome to guide repair.
  • Gene Editing
    • Multiple approaches with varying levels of specificity and potential for off-target effects.
  • RNA modifications
    • Complementary changes to RNA are useful due to reduced defective product outcomes; off-target effects are still a concern.

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Midterm 2: Next Generation Sequencing Methods PDF

Description

This quiz covers key concepts from midterm 2 notes on genome sequencing techniques. Topics include cheaper genome sequencing methodologies, next-generation sequencing (NGS), and the workflow for library preparation. Understand these processes to enhance your knowledge in genetics and molecular biology.

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