Week 5.2 Transcriptomics

Question 2

RNA-Seq

Answer 2

• presence and quantity of RNA
  molecules in a biological sample
• gene expression in sample
• Alternative gene spliced transcripts, exon-intron boundaries
• Post-transcriptional modifications
• Genetic variants such as fusions, indels, SNVs
• Changes or differences in gene expression levels or patterns
• Different populations of RNA subtypes

Question 3

Indirect RNA sequencing

process overview (6)

Answer 3

1. Isolate RNA from samples
2. Fragment DNA into short segments
3. convert RNA fragments into cDNA
4. Ligate sequencing adapters and amplify
5. Perform NGS sequencing
6. Map sequencing reads to transcriptome/genome

Question 4

reverse transcriptase

features

Answer 4

• synthesize DNA from RNA template
• polymerase and nuclease active sites
• retroviruses, prokaryotes, and eukaryotes
• used to extend telomeres in eukaryotes

Question 5

retrovirus

Answer 5

• virus that uses RNA as genomic material
• use reverse transcriptase to convert viral RNA genome into complementary DNA molecule which integrates into whole cell’s genome.
• cell can produce more retrovirus that infects other cells

Question 6

Oxford Nanopore Technologies

Direct RNA sequencing

library preparation process

Answer 6

1. double-stranded Reverse Transcription Adapter (RTA) ligated to RNA through attached complementary sequences (mRNA polyA tail or target specific)
2. Y-shaped adapter ligates with motor protein on RNA strand
3. Motor protein directs RNA through nanopore in 3’5’ direction
4. sequence >30kb read length

Question 7

differential gene expression analysis

Heat map

Answer 7

• row = gene
• column = patient
• Patients grouped together by histologic types of cancer
• red dot = upregulated gene in patient
• green dot = downregulated gene in patient
• patients and genes grouped into clusters = identify differential gene expression patterns associated with different subtypes of cancer

Question 8

Single-cell RNA sequencing

features

Answer 8

• provides expression profile of individual cells
• can identify and characterize transcriptionally distinct subpopulations and states within cell population
• enables detailed unbiased characterization of transcriptional features underlying important phenotypes
• enable accurate characterization of heterogeneity
• understand mechanisms of cancer pathogenesis,
• develop effective treatment strategies
• identify novel targets for immunotherapy and drug development
• current gold standard for defining cell states and phenotypes

Question 9

Single-cell RNA sequencing

process

Answer 9

1. isolate single cells from tissue
2. reverse transcription: individual cells separated into microwells or via emulsion PCR
3. Amplification and sequencing like bulk RNA

Question 10

Single cell sequencing

Reverse Transcription Droplet Method

process

Answer 10

1. Cells encapsulated into droplets in automated machine via microfluidic chip that combines all components with oil
2. Each droplet contains one cell, one gel bead, and reverse transcription reagents
3. 4-part oligos attached to gel bead.
   - sequencing primer
   - 10X barcode unique to gel bead
   - UMI sequence unique to cDNA molecule
   - poly DT sequence to capture cDNA (mRNA polyA tail or target RNA) and functions as primer for reverse transcription.
4. cell is lysed in droplet and undergoes reverse transcription
5. oil is removed and cDNA pooled together for PCR amplification and sequencing.
6. cDNA from the same cell are identified through 10X barcode.
7. number of UMI’s can be used for digital count of cDNA copy to analyze gene expression level

Question 11

Single cell RNA sequencing

Reverse Transcription Droplet Method

features

Answer 11

• 10X Genomics
• data can be used for cellular phenotype classification or new subpopulation identification
• allows detection of rare cell types
• high throughput: 500 to 10,000 cells can be captured per sample from single cell suspension
• need fresh samples
• need to preserve initial relative abundance of MRNA in cell to identify rare transcripts
• requires tissue dissociation and cell isolation

Question 12

Spatial transcriptomics

features

Answer 12

• method for assigning cell types identified by mRNA readouts to their locations in histological section
• used to determine subcellular localization of messenger RNA molecules
• able to capture positional context of transcriptional activity within intact tissues (regions or single cells)
• use intact tissue sections and spatial transcriptome slide (glass slide containing arrays of spatially barcoded oligo probes)

Question 13

Spatial Transcriptomics

process (8)

Answer 13

1. Tissue samples cut into very thin slices, fixed, stained and put on spatial transcriptomic slide
2. undergoes enzymatic permeabilization process so molecules in cell diffuse down to slide
3. mRNA released and binds to barcoded probes
4. Reverse transcription carried out in situ
5. synthesized cDNA contains spatial barcode and UMI provide information about gene expression and location
6. libraries prepared and analyzed by sequencing
7. spatial barcode present within each generated sequence allows data for each mRNA transcript to be mapped back to position on slide.
8. by overlaying picture of tissue with slide, can analyze point of origin for mRNA transcripts within tissue section