Single-cell RNA-Seq data: a (very) short overview · Single-cell RNA-Seq data: a (very) short...
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Single-cell RNA-Seq data: a (very) short overview
March 13, 2020
Dr. ir. Perry D. MoerlandBioinformatics LaboratoryAmsterdam [email protected]
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RNA-Seq
Gene expression => Quite easy (count the reads)Transcript expression => More difficult (but can be done probabilistically)Gene fusion => More difficult (esp. for new fusion events)Splicing => More difficult (esp. for poorly annotated isoforms)
RNA fragmentation
Adaptors + amplification
Sequencing
Map to reference transcriptomeRNA
ACCTAG…CGGTAA…ATGGCA…TGGGAC…TATAGG…
Reverse transcription
Gene A Gene B
Millions of reads
Measured expression is the average over all cell types
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Single-cell RNA-Seq: motivation
Bulk sample gene expression analysis
We have information about all cell types
The signal is mixed with other cell types
Possible in some cases to infer the fraction of the different cell types, but (almost) impossible to infer their actual gene expression profile
Different expression in different cell types in mixed sample
Bimodal expression in a pure sample
Exam
ples
Same average
Same average
gene1 gene1
T1 T2 T1 T2
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Influence of heterogeneity
Signal dilution false negatives
High proportiontumour cells
Low proportiontumour cells
Heterogeneous composition Associated with phenotype
Signal inflation false positives
1
0
0
1
0
0.90.1
Beta value
B
100 1
10 1
00
1 101 1
A
10 1
1
Condition Overall measured signal
Beta value of one probe (0 ≤ B ≤ 1)
0
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Tang et al., Nature Methods (2009)
2009: n=1
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Technological breakthroughs
DIY
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Exponential scaling of single-cell RNA-seq in the past decade
Svensson et al., Nature Methods (2018)
2019Cao et al., Nature
Combinatorial indexing
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scRNA-Seq: workflow
https://en.wikipedia.org/wiki/Single_cell_sequencing
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scRNA-Seq: what can go wrong?
https://en.wikipedia.org/wiki/Single_cell_sequencing
Biased selection of cellsDuplicates
(doublets/multiplets)Broken/dead cells
Dropouts (60-90%)?
Amplification bias
Sequencing errorsRodriques et al., Science (2019): Slide-seq
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Collect RTPool & Remove oil
OilCellsEnzymes
10x BarcodedGel Beads
Single CellGEMs
10x Barcoded
cDNA
10x BarcodedcDNA
Sequencing
Final Library Construct
10x Barcode
read1 read2
P5P7
UMI
Sample Index
Poly(dT)NN
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Typical single-cell RNA-seq analysis workflow
Luecken et al., Molecular Systems Biology (2019)
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Amplification bias: unique molecular identifiers (I)
http://mccarrolllab.com/dropseq/
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QC at cell level
Stegle et al., Nature Reviews Genetics (2015)
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QC at cell level (II)
Luecken et al., Molecular Systems Biology (2019)
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QC at gene level
Lun et al., F1000Research (2016)
Percentage of total counts assigned to the top 50 most highly-abundant features
Does it match expected biology?
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Variability in scRNA-Seq data (I)
Kolodziejczyk et al., Molecular Cell (2015)
Noise
Signal
Extrinsic noise (regulation by transcription factors)vs intrinsIc noise (stochastic bursting/firing, cell cycle)
Overdispersion, batch effects, sequencing depth, GC bias, amplification bias
capture efficiency (starting amount of RNA)
Variability in bulk RNA-SeqAdditional variability in scRNA-seq
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Variability in scRNA-Seq data (II)
Karchenko et al., Nature Methods (2014)
Cell cycle
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NormalizationMethods developed for bulk samples commonly used, but poor fit for scRNA-Seq data…
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Spike-ins (I)
Addition of external controls ERCC spike-ins most widely used, mix consists of 92 mRNAs at
different concentrations Important to add equal amounts to each cell preferably in the
lysis buffer
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Spike-ins (II)
Can be used to model Technical noise Drop-out rates Starting amount of RNA in the cell Data normalization
Two cells from a homogeneous population but with different total mRNA content
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Amplication bias: unique molecular identifiers (II)
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scRNA-Seq: common applications
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Capita selecta
Trade-off between # cells vs. #reads # cells Batch effects, multiplexing Multi-omics
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HashTag Oligonucleotide
Stoekius et al. Gen. Biol., 2018
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https://www.biolegend.com/en-us/totalseq
Antibody DerivedTags
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Demultiplexing HTO
Cells are assigned to belong to a certain HTO (or combination!) based on the sequenced HTO-tags (and cut-offs used…)
Some cells do contain multiple HTO’sMost of these contain HTO’s belonging to the
same subject, i.e. HTO_9/HTO_10 etc. -> Doublets
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HTO tag used tocluster
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Antibody Derived Tags (ADTs)