final genomics

What is the definition of epigenetics?

Epigenetics refers to stable phenotypic changes that occur without alterations in the DNA sequence.

How do epigenetic changes affect gene expression?

They can lead to variations in traits by affecting how genes are expressed without modifying the genetic code.

Are epigenetic changes inheritable, and why is this important?

Yes, they are inheritable, allowing phenotypic characteristics to be passed down through generations.

What is a significant feature of epigenetic changes?

They are reversible, allowing gene expression to change due to environmental factors.

What role do small RNAs play in epigenetic regulation?

They help regulate gene expression by interacting with other molecules in the cell.

What is DNA methylation, and how does it affect gene expression?

DNA methylation involves adding a methyl group, which can inhibit gene expression.

What is the significance of histone modification in epigenetics?

It influences DNA packaging tightness, affecting its accessibility for transcription.

Why is epigenetics considered a crucial field of study?

It explores how stable phenotype changes occur without altering the DNA sequence.

What are the three key mechanisms of epigenetic regulation?

The mechanisms are small RNAs, DNA methylation, and histone modification.

How do epigenetic changes contribute to variations in traits?

They can modify gene expression patterns, leading to observable differences in traits.

What is the purpose of permeabilization in chromatin separation?

Permeabilization allows antibodies and reagents to enter immobilized cells for chromatin analysis.

What is used to achieve permeabilization of cells?

Digitonin, a nonionic detergent, is used to create small holes in the lipid bilayer.

How long should the antibody specific to the target be incubated with chromatin?

The antibody should be incubated overnight at 4°C.

What role does pAG-MNase play in the chromatin analysis process?

pAG-MNase tethers MNase to antibody-bound chromatin for cleavage.

What activates the MNase enzyme during chromatin analysis?

Calcium ions (Ca²⁺) are added to activate MNase.

What happens to the DNA that is cleaved by MNase?

Cleaved DNA fragments diffuse into the supernatant, separating from bulk chromatin.

Why is the magnetic separation of bead-coupled cells important?

It isolates the bulk chromatin from the clipped target DNA for further analysis.

What is the final product of the chromatin separation process?

The final product is a CUT&RUN library for sequencing isolated chromatin fragments.

How does this high-throughput method benefit genomic research?

It streamlines chromatin analysis, enabling high-throughput applications.

What is a nucleosome?

The basic structural unit of chromatin in eukaryotes consisting of DNA wrapped around histone proteins.

How many turns of DNA does a nucleosome contain?

A nucleosome contains a little less than two turns of DNA.

What is the composition of the nucleosome core particle?

Approximately 146 base pairs of DNA wrapped around a histone octamer of eight histone proteins.

Which histone proteins comprise the nucleosome core?

The core histones are H2A, H2B, H3, and H4, with two copies each.

What is heterochromatin?

A highly condensed chromatin structure that is usually transcriptionally silent and located in centromere and telomere regions.

Where is heterochromatin typically found?

Heterochromatin is typically located in or near the centromere and telomere regions.

What is the transcriptional status of heterochromatin?

Heterochromatin is generally transcriptionally silent.

What type of genetic recombination is associated with heterochromatin?

Heterochromatin exhibits low or absent meiotic recombination.

What are the characteristics of euchromatin?

Euchromatin is less condensed, transcriptionally active, and contains a significant number of genes.

Where can euchromatin be found?

Euchromatin is typically found in the chromosomal arms.

What role do histones play in the cell?

Histones prevent DNA tangling and provide protection against DNA damage.

What amino acids are histones rich in?

Histones are rich in lysine (K) and arginine (R) residues.

What are the two main types of histones?

Core histones (H2A, H2B, H3, H4) and linker histones (H1/H5).

How do histones influence gene regulation?

Histones play crucial roles in gene regulation and DNA replication, affecting genetic expression.

What is the difference between heterochromatin and euchromatin concerning meiotic recombination?

Heterochromatin has low recombination, whereas euchromatin exhibits high levels of meiotic recombination.

What is the significance of histones in facilitating DNA functions?

Histones influence the expression and maintenance of genetic information.

What is the Hi-C technique used for in genomic analysis?

Hi-C detects genome-wide chromatin interactions within the cell nucleus.

How does the Hi-C technique combine other genomic methods?

It combines chromosome conformation capture (3C) and next-generation sequencing (NGS).

What are Topologically Associating Domains (TADs)?

TADs are self-interacting genomic regions that interact frequently among themselves.

What indicates the stability of TADs across different cell types?

Their consistent interactions demonstrate their fundamental role in genomic architecture.

What differentiates 'A' compartments from 'B' compartments in the genome?

'A' compartments have open, expression-active chromatin, while 'B' compartments have closed, inactive chromatin.

How does the classification of A/B compartments vary?

It varies between different cell types, reflecting the dynamic chromatin organization.

What methods are used to interpret Hi-C data?

Methods include read mapping, normalization, and correlation analysis.

Name some notable resources for exploring 3D genomes.

Notable resources include Juicebox, WashU Epigenome Browser, and the NIH 4D Nucleome Project.

What functionalities does HiC-Pro provide for Hi-C analysis?

It includes reads mapping, fragment assignment, filtering, quality controls, and ICE normalization.

What is the role of normalization in Hi-C analysis?

Normalization adjusts data to account for biases and ensures accurate comparisons.

What is the primary function of microRNAs (miRNAs)?

miRNAs serve as guide molecules in post-transcriptional gene silencing.

What does the mature miRNA correspond to?

The most prevalent sequence corresponds to the miRNA precursor.

How do miRNAs assist in regulation during development?

They are crucial in regulating development in both animals and plants.

What role do miRNAs play in plants under stress conditions?

miRNAs significantly respond to abiotic and biotic stress in plants.

What is the typical length of small interference RNAs (siRNAs)?

siRNAs are typically 24 nucleotides in length.

What distinguishes siRNAs in terms of abundance?

They are the most abundant type of small RNA.

From what are siRNAs derived?

siRNAs are derived from double-stranded RNAs processed by Dicer-like 3 (DCL3).

What is the main function of siRNAs in genetic regulation?

siRNAs are involved in transcriptional silencing of transposons.

What is the primary epigenetic pathway mediated by siRNAs in plants?

RNA-directed DNA methylation (RdDM) is the primary pathway.

What are 21-22 nt siRNAs and how are they generated?

They include trans-acting siRNAs (tasiRNAs) generated from coding or noncoding transcripts triggered by miRNAs.

What is unique about the arrangement of trans-acting siRNAs (tasiRNAs)?

They are generated in a head-to-tail arrangement starting from a specific nucleotide.

What defines phased siRNAs (phasiRNAs) in their production?

phasiRNAs are produced from protein-coding genes targeted by miRNAs.

How can phasiRNAs function in relation to their configurations?

They can function in either trans or cis configurations.

What is the primary purpose of DESeq2?

DESeq2 is designed for detecting differential gene expression from RNA-seq data.

What are some key methods incorporated in the DESeq2 algorithm?

Normalization, negative binomial modeling, hierarchical modeling, FDR control, and lowly expressed gene filtering.

How does DESeq2 normalize gene read counts?

It normalizes by library size and composition.

Which statistical distribution does DESeq2 use for modeling gene read counts?

DESeq2 uses a negative binomial distribution to model read counts.

What is the role of hierarchical modeling in DESeq2?

Hierarchical modeling stabilizes gene variance during analysis.

What technique does DESeq2 use to control the false discovery rate?

DESeq2 implements the Benjamini-Hochberg procedure for FDR control.

Why does DESeq2 filter lowly expressed genes?

To minimize the number of hypotheses tested in the analysis.

What distribution better represents RNA-seq read count data?

A negative binomial distribution best represents RNA-seq read count data.

Why is a negative binomial distribution preferred in RNA-seq analysis?

It accounts for overdispersion often observed in RNA-seq data.

What is the significance of estimating size factors in DESeq2?

Size factors adjust for differences in library size and composition.

How does DESeq2 assess variability across genes?

By calculating gene-wise dispersion.

What model does DESeq2 fit to the RNA-seq data?

DESeq2 fits a linear generalized linear model (GLM) to the data.

What type of hypothesis testing does DESeq2 utilize?

DESeq2 uses negative binomial generalized linear models for hypothesis testing.

What does DESeq2 aim to control in hypothesis testing?

DESeq2 aims to control the family-wise error rate.

What does the relationship P(false rejection at most one hypothesis) < P(no false rejection) imply?

It describes the control of false rejections among multiple hypotheses.

How should the false rejection rate be adjusted when testing multiple hypotheses?

The rate should be adjusted to α/m at significance level α.

What would be the adjusted p-value cutoff if α is 0.05 for 20,000 genes?

The cutoff would be 0.0000025.

Why can adjusting the false rejection rate be overly conservative?

It can limit the detection of differentially expressed genes with few samples.

What is the false discovery rate (FDR)?

FDR is the rate of type I errors in null hypothesis testing during multiple comparisons.

What is the first step in preparing for the Final Exam according to the exam preparation strategies?

Review the final study guide to understand the key topics and areas of focus.

Why is it important to review the midterm study guide for final exam preparation?

To reinforce foundational knowledge that may be relevant to the final exam.

What materials should students review from Week 1 to Week 4 for the Final Exam?

Lecture slides to revisit important concepts discussed in class.

How can reviewing quizzes help students in exam preparation?

By identifying areas of strength and weakness in their understanding of the material.

What is the role of reviewing homework assignments in final exam preparation?

To ensure comprehension of practical applications of the material.

What is the total point value of the Final Exam?

The Final Exam is worth a total of 180 points.

What does Part I of the Final Exam consist of?

55 multiple choice questions worth 121 points.

What is the time limit for Part I of the Final Exam?

The time limit for Part I is 90 minutes.

How many questions from Weeks 1-6 will be in the multiple-choice section?

There will be 10 questions from Weeks 1-6.

How many essay questions are included in Part II of the Final Exam?

Part II contains 14 essay questions worth 59 points.

What is the time limit for the essay questions section of the Final Exam?

The time limit for essay questions is 180 minutes.

What main topic do the essay questions in the Final Exam cover?

The essay questions cover RNA sequencing (RNA-seq).

What is RNA-seq used for in research?

It analyzes the transcriptome to understand gene regulation across various conditions.

What does an RNA-seq experiment typically involve?

Sequencing RNA transcripts (usually cDNA) to assess gene regulation variation.

What can researchers examine using RNA-seq?

The expression of genes across different samples and conditions.

How does RNA-seq help discover new genes?

It allows the identification of novel genes or transcripts.

What type of events can RNA-seq analyze?

It can study alternative splicing events.

What is one way RNA-seq can contribute to understanding disease?

By contrasting normal versus disease states in gene expression.

What is the typical RNA composition distribution in cells?

rRNA: 85-90%, mRNA: 2-3%, tRNA: 10-12%, other RNAs: 2-3%.

How is RNA-seq library construction different between eukaryotes and prokaryotes?

Eukaryotes use poly-A enrichment or rRNA depletion; prokaryotes focus on rRNA depletion.

Why do prokaryotic mRNA transcripts not contain polyA tails?

Because mRNA transcripts in prokaryotes do not have polyA tails.

What is the initial step in preparing purified CUT&RUN DNA for NGS?

The DNA is subjected to repair and ligation to sequencing adapters.

What role do barcoded primers play during PCR in NGS library preparation?

Barcoded primers facilitate multiplexed sequencing of multiple samples in a single run.