QC of Psen2S4Ter Zebrafish
Steve Pederson
20 January, 2020
Last updated: 2020-01-20
Checks: 6 1
Knit directory: 20170327_Psen2S4Ter_RNASeq/
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Setup
library(ngsReports)
library(tidyverse)
library(magrittr)
library(edgeR)
library(limma)
library(AnnotationHub)
library(ensembldb)
library(scales)
library(pander)
library(cowplot)
library(corrplot)if (interactive()) setwd(here::here())
theme_set(theme_bw())
panderOptions("big.mark", ",")
panderOptions("table.split.table", Inf)
panderOptions("table.style", "rmarkdown")
twoCols <- c(rgb(0.8, 0.1, 0.1), rgb(0.2, 0.2, 0.8))ah <- AnnotationHub() %>%
subset(species == "Danio rerio") %>%
subset(rdataclass == "EnsDb")
ensDb <- ah[["AH74989"]]
grGenes <- genes(ensDb)
grTrans <- transcripts(ensDb)
trLengths <- exonsBy(ensDb, "tx") %>%
width() %>%
vapply(sum, integer(1))
mcols(grTrans)$length <- trLengths[names(grTrans)]
rRnaTrans <- grTrans %>%
subset(grepl("rRNA", tx_biotype)) %>%
mcols() %>%
.[["tx_id_version"]]In order to perform adequate QC, an EnsDb object was obtained for Ensembl release 98 using the AnnotationHub package. This provided the GC content and length for each of the 65,905 transcripts contained in that release.
samples <- read_csv("data/samples.csv") %>%
mutate(reads = str_extract(fqName, "R[12]")) %>%
dplyr::rename(Filename = fqName)
labels <- structure(
samples$sampleID,
names = samples$Filename
)Metadata for each fastq file was also loaded. Reads were provided as paired-end reads, with \(n=4\) samples for each genotype.
Raw Data
Library Sizes
rawFqc <- list.files(
path = "data/0_rawData/FastQC/",
pattern = "zip",
full.names = TRUE
) %>%
FastqcDataList()Library Sizes for the raw, unprocessed dataset ranged between 27,979,654 and 37,144,975 reads.
r1 <- grepl("R1", fqName(rawFqc))
plotReadTotals(rawFqc[r1], labels = labels[r1], barCols = twoCols)
Library Sizes for each sample before any processing was undertaken.
GC Content
As this was a total RNA dataset, GC content will provide a clear marker of the success of rRNA depletion. This was plotted, with all R2 reads showing a far greater spike in GC content at 81%, which is likely due to incomplete rRNA depletion. In particular, the mutant samples appeared most significantly affected raising possibility that overall GC content may be affected in these samples.
gcPlots <- list(
r1 = plotGcContent(
x = rawFqc[r1],
labels = labels[r1],
plotType = "line",
gcType = "Transcriptome",
species = "Drerio"
),
r2 = plotGcContent(
x = rawFqc[!r1],
labels = labels[!r1],
plotType = "line",
gcType = "Transcriptome",
species = "Drerio"
)
)
lg <- get_legend(gcPlots$r2)
plot_grid(
plotlist = list(
r1 = gcPlots$r1 +
ggtitle("R1: GC Distribution", subtitle = c()) +
theme(legend.position = "none"),
r2 = gcPlots$r2 +
ggtitle("R2: GC Distribution", subtitle = c()) +
theme(legend.position = "none"),
lg = lg),
nrow = 1,
rel_widths = c(1,1,1)
)
GC content for R1 and R2 reads. Notably, the R2 reads had clearer spikes in GC content above 70%
rawGC <- getModule(rawFqc, "Per_sequence_GC") %>%
group_by(Filename) %>%
mutate(Freq = Count / sum(Count)) %>%
dplyr::filter(GC_Content > 70) %>%
summarise(Freq = sum(Freq)) %>%
arrange(desc(Freq)) %>%
left_join(samples)
rawGC %>%
ggplot(aes(sampleID, Freq, fill = reads)) +
geom_bar(stat = "identity", position = "dodge") +
facet_wrap(~genotype, scales = "free_x") +
scale_y_continuous(labels = percent) +
scale_fill_manual(values = twoCols) +
labs(x = "Sample", y = "Percent of Total")
Percentages of each library which contain >70% GC. Using the known theoretical distribution, this should be 0.19% of the total library.
Over-represented Sequences
The top 30 Overrepresented sequences were analysed using blastn and most were found to be mitochondrial rRNA sequences.
getModule(rawFqc, "Overrep") %>%
group_by(Sequence, Possible_Source) %>%
summarise(`Found In` = n(), `Highest Percentage` = max(Percentage)) %>%
arrange(desc(`Highest Percentage`), desc(`Found In`)) %>%
ungroup() %>%
dplyr::slice(1:30) %>%
mutate(`Highest Percentage` = percent_format(0.01)(`Highest Percentage`/100)) %>%
pander(
justify = "llrr",
caption = paste(
"*Top", nrow(.),"Overrepresented sequences.",
"The number of samples they were found in is shown,",
"along with the percentage of the most 'contaminated' sample.*"
)
)| Sequence | Possible_Source | Found In | Highest Percentage |
|---|---|---|---|
| GTGGGTTCAGGTAATTAATTTAAAGCTACTTTCGTGTTTGGGCCTCTAGC | No Hit | 12 | 1.72% |
| CTGGGGGAGCGGCCGCCCCGCGGCGCCCCCTCTCGTTCCCGTCTCCGGAG | No Hit | 10 | 1.69% |
| CCGCTGTATTACTCAGGCTGCACTGCAGTGTCTATTCACAGGCGCGATCC | No Hit | 12 | 1.32% |
| GGCCCGGCGCACGTCCAGAGTCGCCGCCGCACACCGCAGCGCATCCCCCC | No Hit | 9 | 1.31% |
| CTCCTGAAAAGGTTGTATCCTTTGTTAAAGGGGCTGTACCCTCTTTAACT | No Hit | 11 | 1.11% |
| GGTTCAGGTAATTAATTTAAAGCTACTTTCGTGTTTGGGCCTCTAGCATC | No Hit | 12 | 1.09% |
| GGGGTGTACGAAGCTGAACTTTTATTCATCTCCCAGACAACCAGCTATTG | No Hit | 12 | 1.07% |
| GGCCCGGCGCACGTCCAGAGTCGCCGCCGCGCACCGCAGCGCATCCCCCC | No Hit | 10 | 1.03% |
| CGAGAGGCTCTAGTTGATATACTACGGCGTAAAGGGTGGTTAAGGAACAA | No Hit | 12 | 1.01% |
| GGGGGAGCGGCCGCCCCGCGGCGCCCCCTCTCGTTCCCGTCTCCGGAGCG | No Hit | 9 | 0.87% |
| CCTCCTTCAAGTATTGTTTCATGTTACATTTTCGTATATTCTGGGGTAGA | No Hit | 12 | 0.82% |
| CCCGCTGTATTACTCAGGCTGCACTGCAGTGTCTATTCACAGGCGCGATC | No Hit | 12 | 0.79% |
| GTTCAGGTAATTAATTTAAAGCTACTTTCGTGTTTGGGCCTCTAGCATCT | No Hit | 12 | 0.79% |
| GGGTTCAGGTAATTAATTTAAAGCTACTTTCGTGTTTGGGCCTCTAGCAT | No Hit | 12 | 0.78% |
| CGGGTCGGGTGGGTGGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGG | No Hit | 12 | 0.73% |
| GGGCCTCTAGCATCTAAAAGCGTATAACAGTTAAAGGGCCGTTTGGCTTT | No Hit | 11 | 0.68% |
| CAGTGGCGTGCGCCTGTAATCCAAGCTACTGGGAGGCTGAGGCTGGCGGA | No Hit | 11 | 0.64% |
| CGGGTCGGGTGGGTAGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGG | No Hit | 12 | 0.57% |
| CTTAGACGACCTGGTAGTCCAAGGCTCCCCCAGGAGCACCATATCGATAC | No Hit | 11 | 0.54% |
| AGCTGGGGAGATCCGCGAGAAGGGCCCGGCGCACGTCCAGAGTCGCCGCC | No Hit | 11 | 0.53% |
| GGCCTCTAGCATCTAAAAGCGTATAACAGTTAAAGGGCCGTTTGGCTTTA | No Hit | 10 | 0.53% |
| CAGCCTATTTAACTTAGGGCCAACCCGTCTCTGTGGCAATAGAGTGGGAA | No Hit | 12 | 0.51% |
| GGGTGGGTGGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGGACGTGG | No Hit | 10 | 0.50% |
| GGGAGCGGCCGCCCCGCGGCGCCCCCTCTCGTTCCCGTCTCCGGAGCGCG | No Hit | 9 | 0.49% |
| CTGGGAGATGAATAAAAGTTCAGCTTCGTACACCCCAAATTAAAAAATTA | No Hit | 10 | 0.48% |
| GCCTATTTAACTTAGGGCCAACCCGTCTCTGTGGCAATAGAGTGGGAAGA | No Hit | 12 | 0.47% |
| GGTCGGGTGGGTGGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGGAC | No Hit | 11 | 0.45% |
| CCCCCGAACCCTTCCAAGCCGAACCGGAGCCGGTCGCGGCGCACCGCCGA | No Hit | 10 | 0.45% |
| GTCGGGTGGGTGGCCGGCATCACCGCGGACCTCGGGCGCCCTTTTGGACG | No Hit | 10 | 0.43% |
| GCCCACTACGACAACGTGTTTTGTAAATTATGATCTTTATTCTCCTGAAA | No Hit | 10 | 0.43% |
Trimming
trimFqc <- list.files(
path = "data/1_trimmedData/FastQC/",
pattern = "zip",
full.names = TRUE
) %>%
FastqcDataList()
trimStats <- readTotals(rawFqc) %>%
dplyr::rename(Raw = Total_Sequences) %>%
left_join(readTotals(trimFqc), by = "Filename") %>%
dplyr::rename(Trimmed = Total_Sequences) %>%
dplyr::filter(grepl("R1", Filename)) %>%
mutate(
Discarded = 1 - Trimmed/Raw,
Retained = Trimmed / Raw
)After adapter trimming between 4.16% and 5.08% of reads were discarded. No other improvement was noted.
Aligned Data
Trimmed reads were:
- aligned to the reference genome using
STAR 2.7.0dand summarised to each gene usingfeatureCounts. These counts were to be used for all gene-level analysis - aligned using kallisto to a modified transcriptome which contained the psen2S4Ter sequence as an ‘alternative’ transcript of psen2. This data was used for genotype checking, and assessing any GC bias in the data. Whilst transcript-level information is included for rRNA genes in the
EnsDbobject, these transcripts are excluded by default from the reference transcriptome provided by Ensembl.
Genotype Checking
cpm(kallisto$counts) %>%
.[c("ENSDART00000006381.8", "psen2S4Ter"),] %>%
as.data.frame() %>%
rownames_to_column("psen2") %>%
pivot_longer(cols = contains("kall"), names_to = "Sample", values_to = "CPM") %>%
mutate(
Sample = basename(Sample),
psen2 = case_when(
psen2 == "psen2S4Ter" ~ "S4Ter",
psen2 != "psen2S4Ter" ~ "WT"
)
) %>%
left_join(samples, by = c("Sample" = "sampleName")) %>%
ggplot(aes(sampleID, CPM, fill = psen2)) +
geom_bar(stat = "identity") +
scale_fill_manual(values = twoCols) +
facet_wrap(~genotype, nrow = 1, scales = "free_x")
CPM obtained for both WT and S4Ter mutant transcripts of psen2
As all samples demonstrated the expected expression patterns, no mislabelling was detected in this dataset.
GC Content
gcInfo <- kallisto$counts %>%
as.data.frame() %>%
rownames_to_column("tx_id") %>%
dplyr::filter(
tx_id != "psen2S4Ter"
) %>%
as_tibble() %>%
pivot_longer(
cols = contains("kall"),
names_to = "sampleName",
values_to = "counts"
) %>%
dplyr::filter(
counts > 0
) %>%
mutate(
sampleName = basename(sampleName),
tx_id_version = tx_id,
tx_id = str_replace(tx_id, "(ENSDART[0-9]+).[0-9]+", "\\1")
) %>%
left_join(
mcols(grTrans) %>% as.data.frame()
) %>%
dplyr::select(
ends_with("id"), sampleName, counts, gc_content, length
) %>%
split(f = .$sampleName) %>%
lapply(function(x){
DataFrame(
gc = Rle(x$gc_content/100, x$counts),
logLen = Rle(log10(x$length), x$counts)
)
}
)
gcSummary <- gcInfo %>%
vapply(function(x){
c(mean(x$gc), sd(x$gc), mean(x$logLen), sd(x$logLen))
}, numeric(4)
) %>%
t() %>%
set_colnames(
c("mn_gc", "sd_gc", "mn_logLen", "sd_logLen")
) %>%
as.data.frame() %>%
rownames_to_column("sampleName") %>%
as_tibble() %>%
left_join(dplyr::filter(samples, reads == "R1")) %>%
dplyr::select(starts_with("sample"), genotype, contains("_"))gcCors <- rawGC %>%
dplyr::filter(reads == "R1") %>%
dplyr::select(starts_with("sample"), genotype, Freq, reads) %>%
left_join(gcSummary) %>%
dplyr::select(Freq, mn_gc, mn_logLen) %>%
cor()The initial QC steps identified an unusually large proportion of reads with >70% GC content. As such an exploration of any residual impacts this may have on the remainder of the library was performed.
A run length encoded (RLE) vector was formed for each sample taking the number of reads for each transcript as the run lengths, and both the GC content and length of each transcript as the values. Transcript lengths were transformed to the log10 scale due to the wide variety of lengths contained in the transcriptome.
From these RLEs, the mean GC and mean length was calculated for each sample, and these values were compared to the proportion of raw reads with > 70% GC, taking these values from the R1 libraries only.
- A correlation of -30% was found between the mean GC content after summarisation to transcript-level counts and the proportion of the original sequence libraries with >70% GC. This makes intuitive sense and suggests that the amount of high-GC reads in the initial libraries, as representative of incomplete rRNA removal, negatively biases the GC composition of the non-ribosomal RNA component of the library.
- A correlation of -80% was found was found between the mean transcript length of the libraries after summarisation to transcript-level counts and the proportion of the original sequence libraries with >70% GC. This is far less obvious mechanistically, but suggests that incomplete rRNA removal also biases the fragmentation or length selection process.
Given the more successful rRNA-removal in the wild-type samples, this dataset is likely to contain significant GC and Length bias which is non-biological in origin.
a <- gcSummary %>%
left_join(rawGC) %>%
dplyr::filter(reads == "R1") %>%
ggplot(aes(Freq, mn_logLen)) +
geom_point(aes(colour = genotype), size = 3) +
geom_smooth(method = "lm") +
scale_shape_manual(values = c(19, 1)) +
labs(
x = "Proportion of initial library with > 70% GC",
y = "Mean log(length)",
colour = "Genotype"
)
b <- gcSummary %>%
left_join(rawGC) %>%
dplyr::filter(reads == "R1") %>%
ggplot(aes(Freq, mn_gc)) +
geom_point(aes(colour = genotype), size = 3) +
geom_smooth(method = "lm") +
scale_shape_manual(values = c(19, 1)) +
scale_y_continuous(labels = percent) +
labs(
x = "Proportion of initial library with > 70% GC",
y = "Mean GC Content",
colour = "Genotype"
)
plot_grid(
a + theme(legend.position = "none"),
b + theme(legend.position = "none"),
get_legend(b),
nrow = 1,
rel_widths = c(4,4,1)
)
Comparison of bias introduced by incomplete rRNA removal. Regression lines are shown along with standard error bands for each comparison.
PCA
transDGE <- kallisto$counts %>%
set_colnames(basename(colnames(.))) %>%
divide_by(kallisto$annotation$Overdispersion) %>%
.[rowSums(.) > 4,] %>%
DGEList(
samples = tibble(
sampleName = colnames(.)
) %>%
left_join(samples) %>%
dplyr::select(sampleName, sample = sampleID, genotype) %>%
distinct(sampleName, .keep_all = TRUE)
) pca <- cpm(transDGE, log = TRUE) %>%
t() %>%
prcomp()pca$x %>%
as.data.frame() %>%
rownames_to_column("sampleName") %>%
left_join(gcSummary) %>%
as_tibble() %>%
left_join(
dplyr::filter(rawGC, reads == "R1")
) %>%
dplyr::select(
PC1, PC2, PC3,
Mean_GC = mn_gc,
Mean_Length = mn_logLen,
Initial_GC70 = Freq,
genotype
) %>%
mutate(genotype = as.numeric(as.factor(genotype))) %>%
cor() %>%
corrplot(
type = "lower",
diag = FALSE,
addCoef.col = 1, addCoefasPercent = TRUE
)
Correlations between the first three principal components and measured variables. Genotypes were converted to an ordered categorical variable for the purposes of visualisation
a <- pca$x %>%
as.data.frame() %>%
rownames_to_column("sampleName") %>%
left_join(
dplyr::filter(rawGC, reads == "R1")
) %>%
as_tibble() %>%
ggplot(aes(PC1, PC2, colour = genotype, size = Freq)) +
geom_point() +
labs(
x = paste0("PC1 (", percent(summary(pca)$importance["Proportion of Variance","PC1"]),")"),
y = paste0("PC2 (", percent(summary(pca)$importance["Proportion of Variance","PC2"]),")"),
size = "Raw GC > 70%",
colour = "Genotype"
)
b <- pca$x %>%
as.data.frame() %>%
rownames_to_column("sampleName") %>%
left_join(gcSummary) %>%
left_join(
dplyr::filter(rawGC, reads == "R1")
) %>%
as_tibble() %>%
ggplot(aes(PC1, mn_gc)) +
geom_point(aes(colour = genotype, size = Freq)) +
geom_smooth(method = "lm") +
scale_y_continuous(labels = percent) +
labs(
x = paste0("PC1 (", percent(summary(pca)$importance["Proportion of Variance","PC1"]),")"),
y = "Mean GC",
size = "Raw GC > 70%",
colour = "Genotype"
)
plot_grid(
plot_grid(
a + theme(legend.position = "none"),
b + theme(legend.position = "none")
),
get_legend(b + theme(legend.position = "bottom")),
nrow = 2,
rel_heights = c(4,1)
)
PCA plot showing genotype, initial GC content > 70% and mean GC content after summarisation to transcript-level.
devtools::session_info()─ Session info ───────────────────────────────────────────────────────────────
setting value
version R version 3.6.2 (2019-12-12)
os Ubuntu 18.04.3 LTS
system x86_64, linux-gnu
ui X11
language en_AU:en
collate en_AU.UTF-8
ctype en_AU.UTF-8
tz Australia/Adelaide
date 2020-01-20
─ Packages ───────────────────────────────────────────────────────────────────
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