Genome Sequencing Midterm 2 Notes

Podcast

Play an AI-generated podcast conversation about this lesson

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?

DNA-RNA interactions are generally more precise. Signup and view all the answers

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

It requires fewer nucleotides to change. Signup and view all the answers

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

To create a phosphodiester bond Signup and view all the answers

What is the ideal sequence read length mentioned?

150 bp Signup and view all the answers

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

It is attached to the phosphate group Signup and view all the answers

What distinguishes the Ion Torrent sequencing method from others?

It is the first platform to sequence DNA without fluorescence Signup and view all the answers

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

To facilitate phosphodiester bond formation Signup and view all the answers

How does the Ion Torrent method detect nucleotide incorporation?

By detecting changes in voltage Signup and view all the answers

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

Denaturation and bridging of the strands Signup and view all the answers

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

0.02 unit change Signup and view all the answers

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

They allow for quicker genome assembly. Signup and view all the answers

How does nanopore sequencing primarily function?

By using a membrane with pores and motor proteins. Signup and view all the answers

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

The modulation of electrical current based on base structure. Signup and view all the answers

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

85% Signup and view all the answers

Which of the following modifications can nanopore sequencing detect?

DNA methylation on cytosines. Signup and view all the answers

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

They may not capture variability present in populations. Signup and view all the answers

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

It helps in identifying genetic causes of diseases. Signup and view all the answers

What is the role of DNA helicase in nanopore sequencing?

To unwind DNA and aid in feeding it through nanopores. Signup and view all the answers

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

To add complimentary nucleotides Signup and view all the answers

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

Identifying genome-wide rare mutations Signup and view all the answers

What role does the primer play in DNA sequencing?

It provides a 3’OH group for DNA polymerase to initiate replication. Signup and view all the answers

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

A view of all SNPs with their statistical significance Signup and view all the answers

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

It reads nucleotide sequences directly from the DNA molecule. Signup and view all the answers

What is the role of bio banks in genetic research?

Distributing and accepting biospecimens Signup and view all the answers

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

To enhance detection of significant SNPs Signup and view all the answers

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

It allows the creation of a consensus sequence to improve accuracy. Signup and view all the answers

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

It decreases after the phosphodiester bond is formed. Signup and view all the answers

Which of the following describes pharmacogenomics?

Study of genetic influences on drug metabolism 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?

Missed potential significant SNPs Signup and view all the answers

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

They separate, trim, and remove adaptor sequences from read data. Signup and view all the answers

Why is real-time sequencing considered advantageous?

It allows for continuous measurement without additional steps. Signup and view all the answers

What is represented by the term PAR in genetic studies?

Proportion of incidence attributable to exposure Signup and view all the answers

What modification is present on nucleotides used in this sequencer?

They are fluorescently labeled on their phosphates. Signup and view all the answers

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

Transparent materials to allow fluorescence measurement. Signup and view all the answers

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

Sanger uses fluorescent nucleotides as chain terminators, whereas Illumina uses reversible terminators. Signup and view all the answers

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

The 3' end of the template strand is at the top, allowing synthesis at the top. Signup and view all the answers

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

They help in introducing all DNTPs at once. Signup and view all the answers

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

The 3' OH of the modified nucleotide is unblocked and the fluorophore is removed. Signup and view all the answers

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

They terminate the DNA chain due to the absence of a 3’ OH. Signup and view all the answers

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

To ensure only one nucleotide is added in each cycle. Signup and view all the answers

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

It ligates the DNA fragments to create longer sequences. Signup and view all the answers

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

Reporter dyes. Signup and view all the answers

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.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

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.