L8 - Transcriptomics Flashcards
What are the two main ‘omics techniques covered in this lecture?
Transcriptomics (RNA-Seq) and Proteomics.
What does RNA-Seq analyze?
RNA-Seq quantifies mRNA expression levels and identifies transcript variants.
What is proteomics?
The study of proteins, including their identification and quantification, often using mass spectrometry.
Why is RNA-Seq considered an ‘open system’?
It sequences RNA without predefined limits, allowing discovery of unknown genes and variants.
What is a ‘closed system’ in transcriptomics?
Techniques like microarrays that only detect predefined genes.
What are the three main steps of RNA-Seq?
RNA extraction and enrichment, sequencing, and data analysis.
How is RNA converted into a form suitable for sequencing?
It is converted into complementary DNA (cDNA).
What sequencing method is typically used in RNA-Seq?
Paired-end sequencing.
What is a FASTQ file?
A file format containing sequence data and quality scores for each read.
What computational challenges does RNA-Seq pose?
Large dataset handling, read mapping, and sequencing bias correction.
What software tools assist in RNA-Seq analysis?
TopHat and Cufflinks.
What virus was studied using RNA-Seq in Bristol?
Adenovirus.
How did adenovirus affect human gene expression in RNA-Seq studies?
Viral transcripts dominated human gene expression over time.
What is the advantage of using RNA-Seq in virology?
It can detect both viral and host responses simultaneously.
What sequencing technology preceded RNA-Seq?
Microarrays.
What is the key limitation of microarrays?
They can only detect known genes and do not identify novel transcripts.
What are the basic steps in viral RNA-Seq experiments?
Infecting cells, extracting RNA, sequencing, and mapping reads.
What was the main finding from adenovirus transcriptomics?
A rapid increase in viral mRNA dominance over human mRNA.
How does RNA-Seq contribute to systems biology?
It provides a holistic view of gene expression interactions.
What does proteomics help determine in infectious disease research?
Functional aspects of gene expression at the protein level.
What is deep sequencing?
High-throughput sequencing providing detailed transcriptomic data.
How does deep sequencing benefit virology?
It reveals viral genetic diversity and host interactions.
What is the significance of FPKM in RNA-Seq?
It normalizes gene expression levels for comparison.
How does nanopore sequencing differ from RNA-Seq?
It sequences RNA directly without converting to cDNA.
What major drawback does nanopore sequencing have?
Higher error rates, particularly insertions and deletions.
What breakthrough did nanopore sequencing achieve for adenovirus?
It revealed over 11,000 transcript variants.
How has RNA-Seq improved over microarrays?
It provides unbiased, quantitative, and variant-specific data.
What is CHiP-Seq used for?
Identifying DNA-protein interactions.
How does whole exome sequencing differ from whole genome sequencing?
It selectively sequences only coding regions.
What is the role of paired-end sequencing?
It sequences both ends of DNA fragments to improve accuracy.
What is the purpose of poly-A selection in RNA-Seq?
To enrich for messenger RNA.
Why is RNA fragmented in RNA-Seq?
To create manageable short reads for sequencing.
How do viruses benefit from alternative splicing?
It allows them to generate diverse protein products.
What is the main advantage of third-generation sequencing?
Long read lengths and direct RNA sequencing capability.
What device enables portable third-generation sequencing?
Nanopore sequencing via a USB-powered device.
What major pandemic virus was analyzed using nanopore sequencing?
SARS-CoV-2.
What is the main computational challenge in RNA-Seq analysis?
Reconstructing full transcripts from short reads.
How does sequencing help detect antiviral drug resistance?
By identifying genetic mutations associated with resistance.
Why is RNA-Seq useful for studying emerging viruses?
It allows unbiased detection of novel viral sequences.
What is de novo assembly in RNA-Seq?
Reconstructing transcripts without a reference genome.
What was a key finding of deep sequencing in HIV studies?
Detection of rare drug-resistant variants.
What type of cells were used in the adenovirus RNA-Seq study?
HeLa cells.
What time points were examined in the adenovirus RNA-Seq study?
0, 8, and 24 hours post-infection.
How many reads were generated per sample in the adenovirus study?
Around 30 million paired-end reads.
What proportion of reads were adenoviral at 24 hours post-infection?
About 80% of mapped reads.
What is the function of TopHat software?
It maps short reads to genomes, considering introns.
What does Cufflinks software do?
It quantifies gene expression levels from RNA-Seq data.
How has RNA-Seq revolutionized virology?
It provides a comprehensive view of viral and host transcriptomes.
What sequencing technology is often used for clinical virus detection?
Nanopore sequencing.
How can sequencing data inform proteomics?
By predicting protein expression from RNA levels.
Why is systems biology important in infectious disease research?
It integrates multiple data types to understand host-pathogen interactions.
How has the cost of sequencing changed over time?
It has drastically decreased, enabling broader use.
What is a key limitation of PCR-based viral genome studies?
It does not provide full transcriptome information.
How do sequencing technologies aid vaccine development?
By identifying viral genetic variation and immune targets.
What challenge does massive sequencing throughput present?
Large computational and storage demands.
What sequencing approach allows rapid identification of new pathogens?
Random deep sequencing of clinical samples.
What technology was crucial for SARS-CoV-2 surveillance?
Third-generation sequencing.
How does transcriptomics aid in understanding viral evolution?
By revealing mutation patterns and splicing diversity.
What type of sequencing does not require prior knowledge of the genome?
De novo sequencing.
How does RNA-Seq contribute to personalized medicine?
By profiling individual gene expression patterns.
What sequencing method can detect epigenetic modifications?
Nanopore sequencing.
What is the purpose of quality scores in FASTQ files?
To assess sequencing accuracy.
How do viruses manipulate host transcription?
By hijacking cellular machinery for their own gene expression.
What does ‘omics integration’ refer to?
Combining transcriptomics, proteomics, and other data.
What is the main advantage of high-throughput sequencing?
Rapid, large-scale genetic analysis.
What biological insight does RNA-Seq provide in infectious diseases?
Host and viral transcriptome interactions.
What is the function of mRNA splicing?
To generate different protein isoforms from the same gene.
Why are sequencing errors a concern in third-generation sequencing?
They can affect variant calling and transcript identification.
What virus family does adenovirus belong to?
Adenoviridae.
What is the primary function of the viral core proteins?
To package and protect the viral genome.
What method is used to analyze viral transcriptomes in great detail?
Nanopore sequencing.
How does sequencing contribute to epidemiology?
By tracking viral mutations and outbreaks.
What makes proteomics essential alongside transcriptomics?
RNA levels do not always correlate directly with protein abundance.
What does ‘fragments per kilobase per million’ (FPKM) measure?
Normalized gene expression in RNA-Seq.
What does ‘deep sequencing’ refer to?
Sequencing at high coverage for detailed analysis.
What is the role of bioinformatics in RNA-Seq?
To process, analyze, and interpret sequencing data.
What major challenge remains in omics research?
Integrating large datasets for meaningful insights.
