Single-Cell RNA Sequencing: From Droplets to a Count Matrix · seed 1 · A4, ink-friendly. The answer key prints on its own page for grown-ups.
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From droplets to a table of cells

Science · Genetics & Evolution · ages 22-23
Name ______________________   Date ____________

  1. Which fields should define a molecule-counting group in this droplet workflow?

    • Gene and UMI, pooled across all droplets
    • Gene, cell barcode and UMI
    • Cell barcode and UMI, pooled across genes

  2. In the bead-based droplet protocol described here, what does a cell barcode identify?

    • The droplet assigned to a read
    • The gene assigned by sequence alignment
    • The original molecule within a gene

  3. UMIs can recover RNA molecules that were never captured and left no detected tagged copy.

    Circle one:   True   False

  4. Some barcodes combine strong markers from two known cell types and have unusually high RNA totals. Which artefact should be investigated first?

    • Ambient RNA adding a few abundant free transcripts
    • A doublet combining RNA from two captured cells
    • UMI collisions merging distinct captured molecules

  5. One captured molecule produces 50 reads with the same gene assignment, cell barcode and UMI. With no tag errors or UMI collisions, how many molecule counts does it contribute?

    • 50
    • 0
    • 1

  6. A gene has zero counts in one cell, although its cell type often expresses it. Which interpretation is justified?

    • The gene is inactive because its cell type fixes its expression
    • Its RNA may be absent or may have escaped detection
    • Its RNA was present because other cells express the gene

  7. Liver-associated RNA occurs in empty droplets and at low levels across unrelated cell types. Which explanation accounts for both observations?

    • Independent transcriptional bursts within each sampled cell
    • Doublets combining intact liver cells with other cell types
    • Ambient RNA entering empty and occupied droplets

  8. After removing suspected doublets, a student says a remaining mixed-profile cluster is proven to be a new cell type. What is missing?

    • Biological evidence for a distinct cell identity
    • A rule that rejects every cell with mixed marker expression
    • A guarantee that clustering removes every technical artefact
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Answer key

For grown-ups. Fold this page away before handing over the rest.

From droplets to a table of cells W1-mt_QCoqY7w_De-s1

  1. Gene, cell barcode and UMI · Grouping must keep both genes and droplets separate. Matching UMI sequences alone do not establish the same molecule.
  2. The droplet assigned to a read · The cell barcode identifies the droplet. Gene assignment uses sequence information; a UMI helps group captured molecules.
  3. False · UMI grouping uses observed tagged reads. Uncaptured RNA leaves no record for that grouping to recover.
  4. A doublet combining RNA from two captured cells · Two-cell capture can combine both marker sets and raise RNA totals. These clues suggest checking doublets, not declaring a certain diagnosis.
  5. 1 · These 50 PCR-derived reads form one molecule group, which contributes one count.
  6. Its RNA may be absent or may have escaped detection · The zero alone cannot separate absent RNA from present RNA missed during capture, reverse transcription or sequencing.
  7. Ambient RNA entering empty and occupied droplets · Free RNA can enter both kinds of droplet. Transcription inside intact cells or doublets alone cannot explain RNA in droplets with no cells.
  8. Biological evidence for a distinct cell identity · Doublet assessment addresses an alternative explanation. It does not establish a new biological identity for the remaining cells.
Worksheet · LightMySky