QC of 7dpf RNAseq dataset
Karissa Barthelson
2021-11-20
Last updated: 2024-11-01
Checks: 7 0
Knit directory:
2021_MPSIIIBvQ96-RNAseq-7dpfLarve/
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| Rmd | 7bb9ce6 | Karissa Barthelson | 2024-11-01 | wflow_publish("analysis/*") |
| Rmd | 6d8b85a | Karissa Barthelson | 2023-03-03 | added adult brain |
| Rmd | 4c30595 | Karissa Barthelson | 2021-11-27 | sat commit |
| html | 4c30595 | Karissa Barthelson | 2021-11-27 | sat commit |
| Rmd | 482cbf7 | Karissa Barthelson | 2021-11-24 | second commit |
Introduction
library(tidyverse)
library(magrittr)
library(readxl)
library(ngsReports)
library(plotly)
library(AnnotationHub)
library(pander)
library(scales)
library(pheatmap)
library(ggpubr)
theme_set(theme_bw())ah <- AnnotationHub() %>%
subset(species == "Danio rerio") %>%
subset(rdataclass == "EnsDb")
ensDb <- ah[["AH83189"]] # for release 101, latest version and the alignment
grTrans <- transcripts(ensDb)
trLengths <- exonsBy(ensDb, "tx") %>%
width() %>%
vapply(sum, integer(1))
mcols(grTrans)$length <- trLengths[names(grTrans)]
gcGene <- grTrans %>%
mcols() %>%
as.data.frame() %>%
dplyr::select(gene_id, tx_id, gc_content, length) %>%
as_tibble() %>%
group_by(gene_id) %>%
summarise(
gc_content = sum(gc_content*length) / sum(length),
length = ceiling(median(length))
)
grGenes <- genes(ensDb)
mcols(grGenes) %<>%
as.data.frame() %>%
left_join(gcGene) %>%
as.data.frame() %>%
DataFrame()meta <- read_excel("data/larvae/meta/naglu_v_Q96_larvae_metadata.xlsx", sheet = 3) %>%
na.omit() %>%
left_join(read_excel("data/larvae/meta/naglu_v_Q96_larvae_metadata.xlsx", sheet = 5) %>%
mutate(temp1 = str_split(temp, pattern = " ")) %>%
mutate(ULN = lapply(temp1, function(x){
x %>%
.[1]
}),
sample_name = lapply(temp1, function(x){
x %>%
.[2]
}),
RIN = lapply(temp1, function(x){
x %>%
.[4]
})
) %>%
unnest() %>%
dplyr::select(ULN, sample_name, RIN) %>%
na.omit() %>%
unique
) %>%
mutate(Genotype = case_when(
`naglu genotype` == "wt" & `psen1 genotype` == "wt" ~ "wt",
`naglu genotype` == "A603fs/A603fs" & `psen1 genotype` == "wt" ~ "MPS-IIIB",
`naglu genotype` == "wt" & `psen1 genotype` == "Q96_K97del/+" ~ "EOfAD-like",
) %>%
factor(levels = c("wt", "MPS-IIIB", "EOfAD-like")))Here, I will assess the quality of the RNA-seq data for the psen1 Q96_K97del/+ vs naglu A603fs/A603fs experiment on zebrafish larvae at 7 days post fertilisation (dpf).
Total RNA was purified from the head end of the individual larvae, while the tail end was used for gDNA extraction and PCR genotyping. The total RNA was DNase treated (to remove any genomic DNA which was carried over from the RNA extraction), then delivered to SAGC for polyA+ library preparation and sequencing using the MGI DNBSEQ technology.
The sequencing was performed over four lanes which were subsequently
merged. This was done using the merge.sh script shown
below.
## Insert the merge files script here
readLines("code/mergeFiles.sh") %>%
cat(sep = "\n")#!/bin/bash
#SBATCH -p batch
#SBATCH -N 1
#SBATCH -n 8
#SBATCH --time=0-01:00:00
#SBATCH --mem=16GB
#SBATCH --mail-type=END
#SBATCH --mail-type=FAIL
#SBATCH --mail-user=karissa.barthelson@adelaide.edu.au
##Params
mkdir /hpcfs/users/a1211024/q96-v-naglu7dpf/temp
FASTDATA1=/hpcfs/users/a1211024/q96-v-naglu7dpf/V350030606
TEMP=/hpcfs/users/a1211024/q96-v-naglu7dpf/temp
FASTOUT=/hpcfs/users/a1211024/q96-v-naglu7dpf/fastq
## Concatenating the F reads
for R1 in ${FASTDATA1}/*_1_R1_001.fastq.gz
do
# Define the other lanes
R2=${R1%_1_R1_001.fastq.gz}_2_R1_001.fastq.gz
R3=${R1%_1_R1_001.fastq.gz}_3_R1_001.fastq.gz
R4=${R1%_1_R1_001.fastq.gz}_4_R1_001.fastq.gz
CATNAME=$(basename ${R1%_1_R1_001.fastq.gz})
echo -e "cat will merge:\t${R1}\n\t${R2}\n\t${R3}\n\t${R4}"
echo -e "New file name will be:\t${TEMP}/${CATNAME}_merged_R1_001.fastq.gz"
cat ${R1} ${R2} ${R3} ${R4} > ${TEMP}/${CATNAME}_merged_R1_001.fastq.gz
done
## Concatenating the R reads
for R1 in ${FASTDATA1}/*_1_R2_001.fastq.gz
do
# Define the other lanes
R2=${R1%_1_R2_001.fastq.gz}_2_R2_001.fastq.gz
R3=${R1%_1_R2_001.fastq.gz}_3_R2_001.fastq.gz
R4=${R1%_1_R2_001.fastq.gz}_4_R2_001.fastq.gz
CATNAME=$(basename ${R1%_1_R2_001.fastq.gz})
echo -e "cat will merge:\t${R1}\n\t${R2}\n\t${R3}\n\t${R4}"
echo -e "New file name will be:\t${TEMP}/${CATNAME}_merged_R2_001.fastq.gz"
cat ${R1} ${R2} ${R3} ${R4} > ${TEMP}/${CATNAME}_merged_R2_001.fastq.gz
done
# ## Move the merged files from temp - fastq
mkdir 01_rawdata
mkdir 01_rawdata/fastq
mv ${TEMP}/*fastq.gz 01_rawdata/fastq
# remove the temp files/dirs
rm ${TEMP}/*.*
rmdir ${TEMP}fastqc: raw data
Here, I will use the ngsReports package to combine and
visualise the fastqc results.
fastqc_raw <- list.files(
path = "data/larvae/fastqc_raw",
pattern = "zip",
recursive = TRUE,
full.names = TRUE) %>%
FastqcDataList()The total number of reads ranged between 60,101,550 and
76,097,954 reads. Note that the number of reads in the
R1 file indeed equals to the number of reads in the
R2 file.
readTotals(fastqc_raw) %>%
mutate(Read = case_when(
grepl(Filename, pattern = "_R1") ~ "R1",
grepl(Filename, pattern = "_R2") ~ "R2"
),
ULN = str_remove(Filename, "_S1_merged.+")
) %>%
left_join(meta) %>%
ggplot(aes(x = ULN, y = Total_Sequences, fill = Read)) +
geom_col(position = "dodge") +
coord_flip() +
scale_fill_viridis_d(end = 0.8) +
facet_wrap(~Genotype, scales = "free_y", ncol = 1, strip.position = "right")
| Version | Author | Date |
|---|---|---|
| 4c30595 | Karissa Barthelson | 2021-11-27 |
The base quality of all the reads also looked good. However,
something a bit strange is going on in the sample 21-015556
file R2 file. Inspecting the boxplot for this file and it looks OK to
me.
plotBaseQuals(fastqc_raw)
| Version | Author | Date |
|---|---|---|
| 4c30595 | Karissa Barthelson | 2021-11-27 |
plotBaseQuals(fastqc_raw[44], plotType = "boxplot")
| Version | Author | Date |
|---|---|---|
| 4c30595 | Karissa Barthelson | 2021-11-27 |
GC Content
All samples have similar GC content. No issues are present.
plotGcContent(
x = fastqc_raw,
plotType = "line",
gcType = "Transcriptome",
species = "Drerio",
usePlotly = TRUE
)Over-repreented seq
No over-represented sequences are present in this dataset.
getModule(fastqc_raw, "Overrep") # A tibble: 0 × 0trimmed data fastQC
The raw fastq. files were then processed with fastp. In
this step, the adaptor sequeces were trimmed from the reads. Then all
length and quality filters were left as default values. Less than 1% of
the reads was discarded, and no observed changes are apparent in the %GC
in the reads.
fastqc_trim <- list.files(path = "data/larvae/fastqc_trim",
pattern = "zip",
recursive = TRUE,
full.names = TRUE) %>%
FastqcDataList()trimStats <- readTotals(fastqc_raw) %>%
dplyr::rename(Raw = Total_Sequences) %>%
left_join(readTotals(fastqc_trim), by = "Filename") %>%
dplyr::rename(Trimmed = Total_Sequences) %>%
mutate(
Discarded = 1 - Trimmed / Raw,
Retained = Trimmed / Raw
)
trimStats %>%
mutate(ULN = str_remove(Filename, "_S1_merged.+")
) %>%
left_join(meta) %>%
unique() %>%
ggplot(aes(y = ULN)) +
geom_col(aes(x = Discarded*100)) +
facet_wrap(~Genotype, scales = "free_y", ncol = 1, strip.position = "right") +
labs(x = "Percentage reads discarded by fastp")
| Version | Author | Date |
|---|---|---|
| 4c30595 | Karissa Barthelson | 2021-11-27 |
plotBaseQuals(fastqc_trim)
| Version | Author | Date |
|---|---|---|
| 4c30595 | Karissa Barthelson | 2021-11-27 |
ggarrange(
plotGcContent(
x = fastqc_raw,
plotType = "line",
gcType = "Transcriptome",
species = "Drerio"
) +
theme(legend.position = "none") +
ggtitle("Before fastp"),
plotGcContent(
x = fastqc_trim,
plotType = "line",
gcType = "Transcriptome",
species = "Drerio"
) +
theme(legend.position = "none")+
ggtitle("After fastp")
) 
| Version | Author | Date |
|---|---|---|
| 4c30595 | Karissa Barthelson | 2021-11-27 |
Aligned QC
The reads were aligned to the GRCz11 genome. The majority of reads were aligned uniquely. S
fastqc_align <- list.files(
path = "data/larvae/fastqc_align",
pattern = "zip",
recursive = TRUE,
full.names = TRUE) %>%
FastqcDataList()list.files("data/larvae/starAlignLog", full.names = TRUE) %>%
.[grepl(x = ., pattern = "Log.final.out")] %>%
ngsReports::plotAlignmentSummary(type = "star") +
scale_fill_viridis_d(end = 0.8) +
theme(legend.position = "right") +
ggtitle("Summary of alignment (STAR)",
subtitle = "In all samples, the majority of reads mapped uniquely to the zebrafish genome.")
| Version | Author | Date |
|---|---|---|
| 4c30595 | Karissa Barthelson | 2021-11-27 |
plotBaseQuals(fastqc_align)
plotGcContent(x = fastqc_align,
plotType = "line",
gcType = "Transcriptome",
species = "Drerio"
) +
theme(legend.position = "none") 
| Version | Author | Date |
|---|---|---|
| 4c30595 | Karissa Barthelson | 2021-11-27 |
Dedup align QC
This dataset was processed with UMIs, which allow PCR duplicates to
be removed. I did this using umi-tools. After
de-duplciation ** reads were retained.
fastqc_align_dedup <- list.files(
path = "data/larvae/fastqc_align_dedup",
pattern = "zip",
recursive = TRUE,
full.names = TRUE) %>%
FastqcDataList()readTotals(fastqc_align) %>%
mutate(align = "raw") %>%
bind_rows(readTotals(fastqc_align_dedup) %>%
mutate(align = "dedup")) %>%
mutate(ULN = str_remove(Filename, "_S1_merged.+")) %>%
left_join(meta) %>%
ggplot(aes(x = ULN, y = Total_Sequences, fill = align)) +
geom_col(position = "dodge") +
coord_flip() +
scale_fill_viridis_d(end = 0.8) +
scale_y_continuous(labels = comma) +
facet_wrap(~Genotype, scales = "free_y", ncol = 1, strip.position = "right")
FeatureCounts summary
The majority of reads are counted i
FC_summary <-
read.delim("data/larvae/featureCounts/counts.out.summary") %>%
set_colnames(colnames(.) %>%
str_remove(pattern = "_S1_merged.Aligned.sortedByCoord.dedup.out.bam") %>%
str_remove(pattern = "X04_dedup.bam.") %>%
str_replace(pattern = "\\.", replacement = "\\-")
)
FC_summary %>%
gather(key = "ULN", value = "NumReads", starts_with("21")) %>%
left_join(meta) %>%
as_tibble() %>%
dplyr::filter(NumReads > 0) %>%
ggplot(aes(y = ULN, x = NumReads, fill = Status)) +
geom_col() +
scale_fill_viridis_d(end = 0.8) +
scale_x_continuous(labels = comma) +
facet_wrap(~Genotype, scales = "free_y", ncol = 1, strip.position = "right")
Conclusion
Data looks of sufficient quality. Proceed to analysis.
sessionInfo()R version 4.3.2 (2023-10-31)
Platform: aarch64-apple-darwin20 (64-bit)
Running under: macOS Sonoma 14.3
Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.11.0
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
time zone: Australia/Adelaide
tzcode source: internal
attached base packages:
[1] stats4 stats graphics grDevices utils datasets methods
[8] base
other attached packages:
[1] ensembldb_2.26.0 AnnotationFilter_1.26.0 GenomicFeatures_1.54.4
[4] AnnotationDbi_1.64.1 Biobase_2.62.0 GenomicRanges_1.54.1
[7] GenomeInfoDb_1.38.8 IRanges_2.36.0 S4Vectors_0.40.2
[10] ggpubr_0.6.0 pheatmap_1.0.12 scales_1.3.0
[13] pander_0.6.5 AnnotationHub_3.10.1 BiocFileCache_2.10.2
[16] dbplyr_2.5.0 plotly_4.10.4 ngsReports_2.4.0
[19] patchwork_1.2.0 BiocGenerics_0.48.1 readxl_1.4.3
[22] magrittr_2.0.3 lubridate_1.9.3 forcats_1.0.0
[25] stringr_1.5.1 dplyr_1.1.4 purrr_1.0.2
[28] readr_2.1.5 tidyr_1.3.1 tibble_3.2.1
[31] ggplot2_3.5.0 tidyverse_2.0.0 workflowr_1.7.1
loaded via a namespace (and not attached):
[1] RColorBrewer_1.1-3 ggdendro_0.2.0
[3] rstudioapi_0.16.0 jsonlite_1.8.8
[5] farver_2.1.1 rmarkdown_2.26
[7] BiocIO_1.12.0 fs_1.6.3
[9] zlibbioc_1.48.2 vctrs_0.6.5
[11] Rsamtools_2.18.0 memoise_2.0.1
[13] RCurl_1.98-1.14 rstatix_0.7.2
[15] S4Arrays_1.2.1 htmltools_0.5.8.1
[17] progress_1.2.3 curl_5.2.1
[19] broom_1.0.5 cellranger_1.1.0
[21] SparseArray_1.2.4 sass_0.4.9
[23] bslib_0.7.0 htmlwidgets_1.6.4
[25] plyr_1.8.9 zoo_1.8-12
[27] cachem_1.0.8 GenomicAlignments_1.38.2
[29] whisker_0.4.1 mime_0.12
[31] lifecycle_1.0.4 pkgconfig_2.0.3
[33] Matrix_1.6-5 R6_2.5.1
[35] fastmap_1.1.1 MatrixGenerics_1.14.0
[37] GenomeInfoDbData_1.2.11 shiny_1.8.1.1
[39] digest_0.6.35 colorspace_2.1-0
[41] ps_1.7.6 rprojroot_2.0.4
[43] crosstalk_1.2.1 RSQLite_2.3.6
[45] labeling_0.4.3 filelock_1.0.3
[47] fansi_1.0.6 timechange_0.3.0
[49] httr_1.4.7 abind_1.4-5
[51] compiler_4.3.2 bit64_4.0.5
[53] withr_3.0.0 backports_1.4.1
[55] BiocParallel_1.36.0 carData_3.0-5
[57] DBI_1.2.2 highr_0.10
[59] ggsignif_0.6.4 biomaRt_2.58.2
[61] MASS_7.3-60.0.1 DelayedArray_0.28.0
[63] rappdirs_0.3.3 rjson_0.2.21
[65] tools_4.3.2 interactiveDisplayBase_1.40.0
[67] httpuv_1.6.15 glue_1.7.0
[69] restfulr_0.0.15 callr_3.7.6
[71] promises_1.3.0 grid_4.3.2
[73] getPass_0.2-4 reshape2_1.4.4
[75] generics_0.1.3 gtable_0.3.4
[77] tzdb_0.4.0 data.table_1.15.4
[79] hms_1.1.3 xml2_1.3.6
[81] car_3.1-2 utf8_1.2.4
[83] XVector_0.42.0 BiocVersion_3.18.1
[85] pillar_1.9.0 later_1.3.2
[87] lattice_0.22-6 rtracklayer_1.62.0
[89] bit_4.0.5 tidyselect_1.2.1
[91] Biostrings_2.70.3 knitr_1.45
[93] git2r_0.33.0 ProtGenerics_1.34.0
[95] SummarizedExperiment_1.32.0 xfun_0.43
[97] matrixStats_1.3.0 DT_0.33
[99] stringi_1.8.3 lazyeval_0.2.2
[101] yaml_2.3.8 codetools_0.2-20
[103] evaluate_0.23 BiocManager_1.30.22
[105] cli_3.6.2 xtable_1.8-4
[107] munsell_0.5.1 processx_3.8.4
[109] jquerylib_0.1.4 Rcpp_1.0.12
[111] png_0.1-8 parallel_4.3.2
[113] XML_3.99-0.16.1 blob_1.2.4
[115] prettyunits_1.2.0 bitops_1.0-7
[117] viridisLite_0.4.2 crayon_1.5.2
[119] rlang_1.1.3 cowplot_1.1.3
[121] KEGGREST_1.42.0