Term
| Outline the steps of Illumina sequence |
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Definition
| 1) Destroy DNA with ultrusound 2) Exonuclease and polymerase to make ends blind 3) Add A and adapters 4) Extract fragments 200-500 bp long with gel 5) PCR with primers to adapters 6) Generate clusters on flow cell with brodge amp 7)Nick and get oosDNA 8) Fluorophores 9)Read and assemble |
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Term
| What could single-cell transcriptomics reveal, that bulk analysis does not? |
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Definition
| Single-cell transcriptomics reveals heterogeneity in gene (co)expression that bulk analysis would not permit. Bulk analysis would detect uniform expression of all three genes. |
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Term
| What are limitations of single cell sequencing? |
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Definition
• Only DNA can be sequenced
• Minimum amount for Illumina > 1 μg
• Amplification required
• Introduction of biases |
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Term
| What constitutes the major part of cellular RNA? |
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Definition
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Term
| Outline main features of microfluids |
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Definition
• reaction volume in nanoliter scale
• 1000-fold less volume
• higher relative template concentration
• more cell-like environment for polymerase
• higher efficiency & less amplification bias |
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Term
| What are the technological advantages of RNA seq? |
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Definition
• technologically easier to amplify than DNA
• average transcript ~ 2-2.5 kb
• PCR less biased than WGA/MDA
• controls |
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Term
| What are the functional advantages of RNA seq? |
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Definition
| • Whole transcriptome essential for functional analysis: |
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Term
| Which steps the single cell rna seq does have on chip? |
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Definition
| 1)Cell loading 2) Capture and wash 3) Lyse 4) cDNA synthesis 5) Preamplification 6)Harvest |
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Term
| Which steps the single cell rna seq does have off chip? |
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Definition
| 1)Clean up 2) Quantify 3) Fragment 4) Adapter ligation 5) PCR library |
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Term
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Definition
| Enzymatic Tagging + Fragmentation in single step! via Tn5 transposase on transposon elements |
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Term
| The main features of circulating tumour cells |
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Definition
• Released from primary tumor
• In the bloodstream (circulation)
• May seed metastasis
• Snapshot of tumor’s genetic composition
• rare population (1-10 / ml)
• accessible to „biopsy“ |
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Term
| What feature of circulating tumor cells can serve as marker? |
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Definition
| detection of upregulated plasma membrane proteins may serve as identifying markers for clinical application |
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Term
| What are the main characteristics of circulating tumor cells? |
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Definition
• downregulation of adherence factors
• migration ability
• downregulation of HLA genes
• escape from immune surveillance |
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Term
| The main features of aposteriory clustering |
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Definition
• unbiased analysis of unique expression
• a posteriori identification of cell types |
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Term
| PINGO: What is the correct order for these events during Illumina sequencing? 1) Excitation and emission of fluorescence 2) Incorporation of labeled nucleotides 3) Denaturation and primer hybridization 4) De-block nucleotides 5) Cluster generation 6) Cleave fluorescence group |
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Definition
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Term
| PINGO: What is the average DNA / RNA content of a single human cell? |
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Definition
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Term
| PINGO: What is the size of the human exome (million bases)? |
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Definition
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Term
| PINGO: Around how much total RNA contains a single human cell? |
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Definition
| 10pg (actually up to 50, this is total, between 2-5% is mRNA) |
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Term
| PINGO: How much picko grams does double genome weight? |
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Definition
| 3.3 - simple, 6.6 - diploid |
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Term
| PINGO: Which of these statements regarding RNA-Seq from single cells are true? |
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Definition
+The terminal transferase activity of the reverse transcriptase allows to hybridize a "template switching oligo" that serves as template to synthesize a defined 3' end of the first strand cDNA
+The small reaction volume in microfluidic devices increases the efficiency of cDNA Synthesis by increasing the concentration of RNA templates |
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Term
| PUNGO: Which statements regarding circulating tumor cells (CTCs) are correct? |
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Definition
+CTCs are of big diagnostic value since they allow to access cells from solid tumors
+One needs markers to enrich CTCs among the many cells in the blood
+CTCs can seed metastases |
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