Quality Control

sce.paul <- readRDS("output/paul_import_sce.RDS")

unfiltered <- sce.paul

For some reason, only one mitochondrial transcripts are available, so we will perform quality control using only the library size and number of detected features. Ideally, we would simply block on the plate of origin to account for differences in processing, but unfortunately, it seems that many plates have a large proportion (if not outright majority) of cells with poor values for both metrics. We identify such plates based on the presence of very low outlier thresholds, for some arbitrary definition of “low”; we then redefine thresholds using information from the other (presumably high-quality) plates.

library(scater)
stats <- perCellQCMetrics(sce.paul) 
qc <- quickPerCellQC(stats, batch=sce.paul$Plate_ID)

# Detecting batches with unusually low threshold values.
lib.thresholds <- attr(qc$low_lib_size, "thresholds")["lower",]
nfeat.thresholds <- attr(qc$low_n_features, "thresholds")["lower",]
ignore <- union(names(lib.thresholds)[lib.thresholds < 100],
    names(nfeat.thresholds)[nfeat.thresholds < 100])

# Repeating the QC using only the "high-quality" batches.
qc2 <- quickPerCellQC(stats, batch=sce.paul$Plate_ID,
    subset=!sce.paul$Plate_ID %in% ignore)
sce.paul <- sce.paul[,!qc2$discard]

We examine the number of cells discarded for each reason.

colSums(as.matrix(qc2))

  low_lib_size low_n_features        discard 
          1695           1781           1783 

We create some diagnostic plots for each metric.

colData(unfiltered) <- cbind(colData(unfiltered), stats)
unfiltered$discard <- qc2$discard
unfiltered$Plate_ID <- factor(unfiltered$Plate_ID)

gridExtra::grid.arrange(
    plotColData(unfiltered, y="sum", x="Plate_ID", colour_by="discard") +
        scale_y_log10() + ggtitle("Total count"),
    plotColData(unfiltered, y="detected", x="Plate_ID", colour_by="discard") +
        scale_y_log10() + ggtitle("Detected features"),
    ncol=1
)

Distribution of each QC metric across cells in the Paul HSC dataset. Each point represents a cell and is colored according to whether that cell was discarded.

saveRDS(sce.paul,"output/paul_quality_control_sce.RDS")

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Packages

package	version	date
Rcpp	1.0.6	2021-01-16
rsvd	1.0.3	2020-07-15
lattice	0.20-41	2020-06-07
digest	0.6.27	2020-10-25
assertthat	0.2.1	2020-07-15
SingleCellExperiment	1.12.0	2020-10-28
utf8	1.1.4	2020-07-15
R6	2.5.0	2020-10-29
GenomeInfoDb	1.26.2	2020-12-09
stats4	4.0.1	2020-06-07
evaluate	0.14	2020-06-15
highr	0.8	2020-07-15
ggplot2	3.3.3	2020-12-31
pillar	1.6.0	2021-04-14
sparseMatrixStats	1.2.0	2020-10-28
utils	4.0.1	2020-06-07
zlibbioc	1.36.0	2020-10-29
rlang	1.1.1	2023-07-17
irlba	2.3.3	2020-07-15
S4Vectors	0.28.1	2020-12-10
Matrix	1.2-18	2020-06-07
BiocNeighbors	1.8.2	2020-12-08
BiocParallel	1.24.1	2020-11-07
stringr	1.4.0	2020-07-15
RCurl	1.98-1.2	2020-07-15
munsell	0.5.0	2020-07-15
beachmat	2.6.4	2020-12-21
DelayedArray	0.16.0	2020-10-28
compiler	4.0.1	2020-06-07
vipor	0.4.5	2020-07-15
BiocSingular	1.6.0	2020-10-28
xfun	0.39	2023-07-17
pkgconfig	2.0.3	2020-07-15
stats	4.0.1	2020-06-07
BiocGenerics	0.36.0	2020-10-28
ggbeeswarm	0.6.0	2020-07-16
tidyselect	1.1.0	2020-07-15
SummarizedExperiment	1.20.0	2020-10-28
tibble	3.1.1	2021-04-19
gridExtra	2.3	2020-07-15
GenomeInfoDbData	1.2.4	2020-11-03
IRanges	2.24.1	2020-12-13
matrixStats	0.57.0	2020-09-26
grDevices	4.0.1	2020-06-07
viridisLite	0.3.0	2020-06-15
fansi	0.4.2	2021-01-16
crayon	1.4.1	2021-02-09
dplyr	1.0.5	2021-03-06
withr	2.4.2	2021-04-19
bitops	1.0-6	2020-07-15
grid	4.0.1	2020-06-07
gtable	0.3.0	2020-07-15
lifecycle	1.0.0	2021-02-16
DBI	1.1.1	2021-01-16
git2r	0.28.0	2021-01-11
magrittr	2.0.1	2020-11-18
datasets	4.0.1	2020-06-07
scales	1.1.1	2020-07-16
cli	2.5.0	2021-04-27
stringi	1.5.3	2020-09-10
scuttle	1.0.4	2020-12-18
farver	2.0.3	2020-07-15
XVector	0.30.0	2020-10-29
viridis	0.5.1	2020-07-17
scater	1.18.3	2020-11-09
DelayedMatrixStats	1.12.2	2021-01-13
ellipsis	0.3.1	2020-07-15
graphics	4.0.1	2020-06-07
generics	0.1.0	2020-11-01
vctrs	0.3.6	2020-12-18
cowplot	1.1.1	2020-12-31
base	4.0.1	2020-06-07
tools	4.0.1	2020-06-07
Biobase	2.50.0	2020-10-28
glue	1.4.2	2020-08-28
beeswarm	0.2.3	2020-07-15
purrr	0.3.4	2020-07-15
MatrixGenerics	1.2.0	2020-10-28
parallel	4.0.1	2020-06-07
colorspace	2.0-0	2020-11-12
GenomicRanges	1.42.0	2020-10-28
knitr	1.30	2020-09-23
methods	4.0.1	2020-06-07

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System Information

	systemInfo
version	R version 4.0.1 (2020-06-06)
platform	x86_64-apple-darwin17.0 (64-bit)
locale	en_CA.UTF-8
OS	macOS 10.16
UI	X11

Scikick Configuration

cat scikick.yml

### Scikick Project Workflow Configuration File

# Directory where Scikick will store all standard notebook outputs
reportdir: report

# --- Content below here is best modified by using the Scikick CLI ---

# Notebook Execution Configuration (format summarized below)
# analysis:
#  first_notebook.Rmd:
#  second_notebook.Rmd: 
#  - first_notebook.Rmd     # must execute before second_notebook.Rmd
#  - functions.R            # file is used by second_notebook.Rmd
#
# Each analysis item is executed to generate md and html files, E.g.:
# 1. <reportdir>/out_md/first_notebook.md
# 2. <reportdir>/out_html/first_notebook.html
analysis: !!omap
- index.Rmd:
- notebooks/nestorowa/import.Rmd:
- notebooks/nestorowa/quality_control.Rmd:
  - notebooks/nestorowa/import.Rmd
- notebooks/nestorowa/normalization.Rmd:
  - notebooks/nestorowa/quality_control.Rmd
- notebooks/nestorowa/further_exploration.Rmd:
  - notebooks/nestorowa/normalization.Rmd
- notebooks/grun/import.Rmd:
- notebooks/grun/quality_control.Rmd:
  - notebooks/grun/import.Rmd
- notebooks/grun/normalization.Rmd:
  - notebooks/grun/quality_control.Rmd
- notebooks/grun/further_exploration.Rmd:
  - notebooks/grun/normalization.Rmd
- notebooks/paul/import.Rmd:
- notebooks/paul/quality_control.Rmd:
  - notebooks/paul/import.Rmd
- notebooks/paul/normalization.Rmd:
  - notebooks/paul/quality_control.Rmd
- notebooks/paul/further_exploration.Rmd:
  - notebooks/paul/normalization.Rmd
- notebooks/merged/merge.Rmd:
  - notebooks/grun/quality_control.Rmd
  - notebooks/paul/quality_control.Rmd
  - notebooks/nestorowa/normalization.Rmd
- notebooks/merged/combined_analysis.Rmd:
  - notebooks/merged/merge.Rmd
version_info:
  snakemake: 6.0.2
  ruamel.yaml: 0.16.12
  scikick: 0.2.1
# Optional site theme customization
output:
  BiocStyle::html_document:
    code_folding: hide
    theme: readable
    toc_float: true
    toc: true
    number_sections: false
    toc_depth: 5
    self_contained: true

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