From b6981cdad469d0edb524917d51f467b1ed90acc2 Mon Sep 17 00:00:00 2001
From: Tom Winkler <t.winkler@uni-koeln.de>
Date: Thu, 26 Sep 2024 10:25:44 +0200
Subject: [PATCH] adjusted paths to ARC structure
---
assays/BSA_RNAseq/adapters/custom_adapters.fa | 24 +++
assays/BSA_RNAseq/adapters/readme.txt | 3 +
assays/BSA_RNAseq/isa.assay.xlsx | Bin 9293 -> 9292 bytes
assays/BSA_WGS/isa.assay.xlsx | Bin 9311 -> 9310 bytes
.../isa.assay.xlsx | Bin 9277 -> 9276 bytes
.../isa.assay.xlsx | Bin 10101 -> 10100 bytes
assays/isoform_sequencing/isa.assay.xlsx | Bin 9324 -> 9323 bytes
isa.investigation.xlsx | Bin 8417 -> 8416 bytes
.../BSA_parent_betalain_quant/isa.study.xlsx | Bin 12584 -> 12583 bytes
.../Bulk_segregant_analysis/isa.study.xlsx | Bin 10586 -> 10585 bytes
studies/additional_data/README.md | 0
studies/additional_data/isa.study.xlsx | Bin 0 -> 7504 bytes
studies/additional_data/protocols/.gitkeep | 0
studies/additional_data/resources/.gitkeep | 0
.../resources/color_pathway_genes.bed | 36 +++++
.../resources/color_pathway_genes.csv | 30 ++++
studies/isoform_sequencing/isa.study.xlsx | Bin 10164 -> 10163 bytes
workflows/BSA/RNAseq/QC.sh | 18 +--
workflows/BSA/RNAseq/adapter_trimming.sh | 4 +-
workflows/BSA/RNAseq/index_STAR.sh | 6 +-
workflows/BSA/RNAseq/run_STAR.sh | 4 +-
workflows/BSA/RNAseq/run_kallisto.sh | 4 +-
workflows/BSA/WGS/combined_filter.sh | 6 +-
workflows/BSA/WGS/map_reads.sh | 6 +-
workflows/BSA/betalain_quantification.Rmd | 80 +++++-----
workflows/BSA/bsa_and_plotting.R | 102 ++++++------
workflows/BSA/read_count_analysis.Rmd | 16 +-
.../BSA/read_count_analysis_from_bam.Rmd | 69 ++++-----
workflows/BSA/snpEff_analysis.Rmd | 146 +++++++++---------
workflows/genome_polishing/helper_script.R | 12 +-
.../process_nextpolish_output.sh | 14 +-
.../remove_ambiguous_bases.sh | 7 +-
workflows/genome_polishing/run_nextpolish.sh | 8 +-
workflows/genome_polishing/unpackSRA.sh | 18 +--
34 files changed, 341 insertions(+), 272 deletions(-)
create mode 100644 assays/BSA_RNAseq/adapters/custom_adapters.fa
create mode 100644 assays/BSA_RNAseq/adapters/readme.txt
create mode 100644 studies/additional_data/README.md
create mode 100644 studies/additional_data/isa.study.xlsx
create mode 100644 studies/additional_data/protocols/.gitkeep
create mode 100644 studies/additional_data/resources/.gitkeep
create mode 100644 studies/additional_data/resources/color_pathway_genes.bed
create mode 100644 studies/additional_data/resources/color_pathway_genes.csv
diff --git a/assays/BSA_RNAseq/adapters/custom_adapters.fa b/assays/BSA_RNAseq/adapters/custom_adapters.fa
new file mode 100644
index 0000000..c6da458
--- /dev/null
+++ b/assays/BSA_RNAseq/adapters/custom_adapters.fa
@@ -0,0 +1,24 @@
+>read1_1/1
+AGATCGGAAGAGCACACGTCTGAACTCCAGTCA
+>read1_2/1
+AGATCGGAAGAGCACACGTCTGAAC
+>read1_3/1
+TGGAATTCTCGGGTGCCAAGG
+>read1_4/1
+AGATCGGAAGAGCACACGTCT
+>read1_5/1
+CTGTCTCTTATACACATCT
+>read1_6/1
+AGATGTGTATAAGAGACAG
+>read2_1/2
+AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
+>read2_2/2
+AGATCGGAAGAGCGTCGTGTAGGGA
+>read2_3/2
+TGGAATTCTCGGGTGCCAAGG
+>read2_4/2
+AGATCGGAAGAGCACACGTCT
+>read2_5/2
+CTGTCTCTTATACACATCT
+>read2_6/2
+AGATGTGTATAAGAGACAG
diff --git a/assays/BSA_RNAseq/adapters/readme.txt b/assays/BSA_RNAseq/adapters/readme.txt
new file mode 100644
index 0000000..a029aca
--- /dev/null
+++ b/assays/BSA_RNAseq/adapters/readme.txt
@@ -0,0 +1,3 @@
+## raw_data/BSA_rnaseq/adapters/
+
+Sequencing adapters used for reads trimming
diff --git a/assays/BSA_RNAseq/isa.assay.xlsx b/assays/BSA_RNAseq/isa.assay.xlsx
index 2d21f88fb855a54a1f3982155f0bb99249397390..399f4b8a40fb6354edb648cda747f4dc1d558145 100644
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diff --git a/assays/BSA_WGS/isa.assay.xlsx b/assays/BSA_WGS/isa.assay.xlsx
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diff --git a/assays/betalain_LCMS_quantification/isa.assay.xlsx b/assays/betalain_LCMS_quantification/isa.assay.xlsx
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diff --git a/assays/isoform_sequencing/isa.assay.xlsx b/assays/isoform_sequencing/isa.assay.xlsx
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literal 0
HcmV?d00001
diff --git a/studies/additional_data/protocols/.gitkeep b/studies/additional_data/protocols/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/studies/additional_data/resources/.gitkeep b/studies/additional_data/resources/.gitkeep
new file mode 100644
index 0000000..e69de29
diff --git a/studies/additional_data/resources/color_pathway_genes.bed b/studies/additional_data/resources/color_pathway_genes.bed
new file mode 100644
index 0000000..c4ee581
--- /dev/null
+++ b/studies/additional_data/resources/color_pathway_genes.bed
@@ -0,0 +1,36 @@
+chr start end strand gene_id transcript_id Gene Pathway
+Scaffold_1 7523181 7527088 - AHp000674 AHp000674.1 AhCYP76AD5 Betalain
+Scaffold_1 28687404 28688868 - AHp001663 AHp001663.1 AhBetanidin5GT Betalain
+Scaffold_2 6806355 6807984 + AHp003152 AHp003152.1 F3-H_3 Flavonoid
+Scaffold_2 6806355 6807984 + AHp003152 AHp003152.2 F3-H_3 Flavonoid
+Scaffold_2 31518210 31522095 + AHp004305 AHp004305.1 CHS Flavonoid
+Scaffold_2 31518210 31522095 + AHp004305 AHp004305.2 CHS Flavonoid
+Scaffold_3 14652200 14653652 + AHp005940 AHp005940.1 AhBetanidin6GT Betalain
+Scaffold_3 29507485 29509926 + AHp006454 AHp006454.1 F3H Flavonoid
+Scaffold_4 10226509 10227946 - AHp007219 AHp007219.1 AhcDOPA5GT Betalain
+Scaffold_5 9470955 9474986 - AHp008991 AHp008991.1 FLS Flavonoid
+Scaffold_5 9470955 9474986 - AHp008991 AHp008991.2 FLS Flavonoid
+Scaffold_5 19537497 19543306 + AHp009303 AHp009303.1 DFR Flavonoid
+Scaffold_6 3461614 3469253 - AHp009962 AHp009962.1 CHI1 Flavonoid
+Scaffold_6 13877397 13882497 + AHp010386 AHp010386.1 AhDODAα2 Betalain
+Scaffold_8 3308349 3313908 + AHp012752 AHp012752.1 PAL_1 Flavonoid
+Scaffold_8 7925628 7930892 - AHp013217 AHp013217.1 C4H_1 Flavonoid
+Scaffold_8 7925628 7930892 - AHp013217 AHp013217.2 C4H_1 Flavonoid
+Scaffold_9 12115220 12125730 - AHp014409 AHp014409.1 4CL_1 Flavonoid
+Scaffold_10 21762620 21764924 - AHp016530 AHp016530.1 AhMYB3 Betalain
+Scaffold_10 21780564 21783050 - AHp016531 AHp016531.1 AhMYB4 Betalain
+Scaffold_11 16155424 16159543 + AHp017409 AHp017409.1 LAR Flavonoid
+Scaffold_11 17204504 17209738 - AHp017497 AHp017497.1 F3-H_1 Flavonoid
+Scaffold_14 12286837 12295353 + AHp020962 AHp020962.1 4CL_2 Flavonoid
+Scaffold_15 1098208 1100359 + AHp021795 AHp021795.1 ANS Flavonoid
+Scaffold_15 2886482 2891165 - AHp021980 AHp021980.1 PAL_2 Flavonoid
+Scaffold_15 4193988 4197809 + AHp022120 AHp022120.1 F3-H_4 Flavonoid
+Scaffold_15 4211064 4214362 + AHp022122 AHp022122.1 F3-H_2 Flavonoid
+Scaffold_15 4227367 4229108 - AHp022123 AHp022123.1 F3-H_5 Flavonoid
+Scaffold_15 7959672 7965298 + AHp022382 AHp022382.1 C4H_3 Flavonoid
+Scaffold_15 7959672 7965298 + AHp022382 AHp022382.2 C4H_3 Flavonoid
+Scaffold_15 8003999 8008333 + AHp022384 AHp022384.1 C4H_2 Flavonoid
+Scaffold_16 988733 991447 + AHp022773 AHp022773.1 AhMYB2 Betalain
+Scaffold_16 988733 991447 + AHp022773 AHp022773.2 AhMYB2 Betalain
+Scaffold_16 5231548 5239058 - AHp023147 AHp023147.1 AhDODAα1 Betalain
+Scaffold_16 5301961 5305973 + AHp023148 AHp023148.1 AhCYP76AD2 Betalain
diff --git a/studies/additional_data/resources/color_pathway_genes.csv b/studies/additional_data/resources/color_pathway_genes.csv
new file mode 100644
index 0000000..a427fab
--- /dev/null
+++ b/studies/additional_data/resources/color_pathway_genes.csv
@@ -0,0 +1,30 @@
+Gene,Pathway,Gene_id
+PAL_1,Flavonoid,AHp012752
+PAL_2,Flavonoid,AHp021980
+C4H_1,Flavonoid,AHp013217
+C4H_2,Flavonoid,AHp022384
+C4H_3,Flavonoid,AHp022382
+4CL_1,Flavonoid,AHp014409
+4CL_2,Flavonoid,AHp020962
+CHS,Flavonoid,AHp004305
+CHI1,Flavonoid,AHp009962
+F3-H_1,Flavonoid,AHp017497
+F3-H_2,Flavonoid,AHp022122
+F3-H_3,Flavonoid,AHp003152
+F3-H_4,Flavonoid,AHp022120
+F3-H_5,Flavonoid,AHp022123
+DFR,Flavonoid,AHp009303
+F3H,Flavonoid,AHp006454
+FLS,Flavonoid,AHp008991
+LAR,Flavonoid,AHp017409
+ANS,Flavonoid,AHp021795
+AhCYP76AD2,Betalain,AHp023148
+AhCYP76AD5,Betalain,AHp000674
+AhDODAα2,Betalain,AHp010386
+AhDODAα1,Betalain,AHp023147
+AhcDOPA5GT,Betalain,AHp007219
+AhBetanidin5GT,Betalain,AHp001663
+AhBetanidin6GT,Betalain,AHp005940
+AhMYB2,Betalain,AHp022773
+AhMYB3,Betalain,AHp016530
+AhMYB4,Betalain,AHp016531
diff --git a/studies/isoform_sequencing/isa.study.xlsx b/studies/isoform_sequencing/isa.study.xlsx
index 60867b92cbe45252333f78b49c485bd39a3e02b2..48af51fa63c14650219e5e214e849d894b1120dd 100644
GIT binary patch
delta 701
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delta 702
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diff --git a/workflows/BSA/RNAseq/QC.sh b/workflows/BSA/RNAseq/QC.sh
index 64db677..947a4ff 100644
--- a/workflows/BSA/RNAseq/QC.sh
+++ b/workflows/BSA/RNAseq/QC.sh
@@ -22,8 +22,8 @@ GTFIN=polished_genome_annotation/annotation/Ahypochondriacus_2.2_polished_correc
# Quality control
# Initialize the output directory
-QCIN=raw_data/BSA_rnaseq/
-QCOUT=raw_data/BSA_rnaseq/fastqc
+QCIN=assays/BSA_RNAseq/dataset
+QCOUT=assays/BSA_RNAseq/fastqc
mkdir -p $QCOUT
# run quality control fastqc
@@ -32,14 +32,14 @@ fastqc -t 10 -o $QCOUT "$QCIN"*P.fq.gz
# Quality control
# Initialize the output directory
-QCIN=raw_data/BSA_rnaseq/
-QCOUT=raw_data/BSA_rnaseq/fastqc
+QCIN=assays/BSA_RNAseq/
+QCOUT=assays/BSA_RNAseq/fastqc
mkdir -p $QCOUT
# run quality control fastqc
fastqc -t 10 -o $QCOUT "$QCIN"*P.fq.gz
-cp -r $QCOUT data/BSA/RNAseq/STAR_flower_mappings/QC/
+cp -r $QCOUT runs/BSA/RNAseq/STAR_flower_mappings/QC/
module load samtools/1.13
@@ -53,11 +53,11 @@ GTFQM=/scratch/twinkle1/temp.gtf
sed 's/CDS/exon/' $GTFIN > $GTFQM
# define input files
-QMIN=data/BSA/RNAseq/STAR_flower_mappings/
+QMIN=runs/BSA/RNAseq/STAR_flower_mappings/
# define output directory
-QMOUT=data/BSA/RNAseq/STAR_flower_mappings/QC/qualimap
+QMOUT=runs/BSA/RNAseq/STAR_flower_mappings/QC/qualimap
# create main output directory
mkdir -p $QMOUT
@@ -85,8 +85,8 @@ done
rm $GTFQM
# run multiqc to combine the results from fastqc and qualimap into a single report
-MULTIQCOUT=data/BSA/RNAseq/STAR_flower_mappings/multiqc
-MULTIQCIN=data/BSA/RNAseq/STAR_flower_mappings/QC/
+MULTIQCOUT=runs/BSA/RNAseq/STAR_flower_mappings/multiqc
+MULTIQCIN=runs/BSA/RNAseq/STAR_flower_mappings/QC/
mkdir -p $MULTIQCOUT
diff --git a/workflows/BSA/RNAseq/adapter_trimming.sh b/workflows/BSA/RNAseq/adapter_trimming.sh
index 15d5ab7..2821fec 100644
--- a/workflows/BSA/RNAseq/adapter_trimming.sh
+++ b/workflows/BSA/RNAseq/adapter_trimming.sh
@@ -20,7 +20,7 @@ module load trimmomatic/0.39
# run this part as array job
# create array of read fastq files (R1 only):
-SOURCE_DIR=raw_data/BSA_rnaseq/
+SOURCE_DIR=assays/BSA_RNAseq/dataset/
FILES=("$SOURCE_DIR"/*R1.fastq.gz)
# run trimmomatic, use 6 threads, taking advantage of the baseout function to name output files
@@ -32,4 +32,4 @@ java -jar $TRIMMOMATIC/trimmomatic.jar PE \
"${FILES["${SLURM_ARRAY_TASK_ID}"]}" \
"${FILES["${SLURM_ARRAY_TASK_ID}"]/R1.fastq.gz/R2.fastq.gz}" \
-baseout "${FILES["${SLURM_ARRAY_TASK_ID}"]/R1.fastq.gz/trimmed.fq.gz}" \
- ILLUMINACLIP:raw_data/BSA_rnaseq/adapters/custom_adapters.fa:2:30:10
+ ILLUMINACLIP:assays/BSA_RNAseq/adapters/custom_adapters.fa:2:30:10
diff --git a/workflows/BSA/RNAseq/index_STAR.sh b/workflows/BSA/RNAseq/index_STAR.sh
index cb8606a..6d12c7f 100644
--- a/workflows/BSA/RNAseq/index_STAR.sh
+++ b/workflows/BSA/RNAseq/index_STAR.sh
@@ -18,10 +18,10 @@ module load star/2.7.8a
# more specific settings: use the polished, softmasked reference assembly
# sjdbOverhang dependend on input read length
# as SJDB file, use the newly generated braker2 protein gtf file
-REFGENOME=polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta
-SJDBFILE=polished_genome_annotation/annotation/Ahypochondriacus_2.2_polished_corrected.gtf
+REFGENOME=runs/polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta
+SJDBFILE=runs/polished_genome_annotation/annotation/Ahypochondriacus_2.2_polished_corrected.gtf
-mkdir -p data/BSA/RNAseq/STAR_flower_index
+mkdir -p runs/BSA/RNAseq/STAR_flower_index
STAR --runThreadN 8 \
--runMode genomeGenerate \
diff --git a/workflows/BSA/RNAseq/run_STAR.sh b/workflows/BSA/RNAseq/run_STAR.sh
index 1064306..099d4e6 100644
--- a/workflows/BSA/RNAseq/run_STAR.sh
+++ b/workflows/BSA/RNAseq/run_STAR.sh
@@ -12,9 +12,9 @@
module load star/2.7.8a
# create array of read fastq files (R1 only):
-SOURCE_DIR=raw_data/BSA_rnaseq/
+SOURCE_DIR=assays/BSA_RNAseq/dataset/
FILES=("$SOURCE_DIR"/*_1P.fq.gz)
-OUTPUTDIR=data/BSA/RNAseq/STAR_flower_mappings
+OUTPUTDIR=runs/BSA/RNAseq/STAR_flower_mappings
# change directory of outprefix
OUTPREFIX1=("${FILES["${SLURM_ARRAY_TASK_ID}"]/$SOURCE_DIR/$OUTPUTDIR}")
diff --git a/workflows/BSA/RNAseq/run_kallisto.sh b/workflows/BSA/RNAseq/run_kallisto.sh
index ae124fb..88243f0 100644
--- a/workflows/BSA/RNAseq/run_kallisto.sh
+++ b/workflows/BSA/RNAseq/run_kallisto.sh
@@ -23,9 +23,9 @@
##### Perform quantification
# create array of read fastq files (R1 only):
-SOURCE_DIR=raw_data/flower_color_mapping/
+SOURCE_DIR=assays/BSA_RNAseq/dataset/flower_color_mapping/
FILES=("$SOURCE_DIR"AM*_1P.fq.gz)
-OUTDIR=data/flower_color_mapping/kallisto_quant/
+OUTDIR=runs/flower_color_mapping/kallisto_quant/
TISSUE_NAMES=("AM_00331_gf" "AM_00331_rf" "AM_00332_gf" "AM_00332_rf")
mkdir -p $OUTDIR
diff --git a/workflows/BSA/WGS/combined_filter.sh b/workflows/BSA/WGS/combined_filter.sh
index 707a9bd..78e6a17 100644
--- a/workflows/BSA/WGS/combined_filter.sh
+++ b/workflows/BSA/WGS/combined_filter.sh
@@ -12,13 +12,13 @@
module load samtools/1.13
-REFERENCE=polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta
+REFERENCE=runs/polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta
PROVIDER=CCG
-OUTDIR=data/BSA/wgs/vcf
+OUTDIR=runs/BSA/wgs/vcf
mkdir -p $OUTDIR
-ALLSAMP=$(for i in data/BSA/wgs/bam_files/gvcf/AM_00*.vcf; do echo -V $i;done)
+ALLSAMP=$(for i in runs/BSA/wgs/bam_files/gvcf/AM_00*.vcf; do echo -V $i;done)
$MYUTIL/tools/gatk-4.1.7.0/gatk --java-options "-Xmx48G" \
CombineGVCFs \
diff --git a/workflows/BSA/WGS/map_reads.sh b/workflows/BSA/WGS/map_reads.sh
index ad2bcf2..3a1d2a8 100644
--- a/workflows/BSA/WGS/map_reads.sh
+++ b/workflows/BSA/WGS/map_reads.sh
@@ -17,15 +17,15 @@ module load bwamem2/2.0_gnu
module load samtools/1.13
-REFERENCE=polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta
+REFERENCE=runs/polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta
#bwa-mem2 index $REFERENCE
PROVIDER=CCG
-INPUTPATH=raw_data/BSA_wgs/ #
-OUTPUTPATH=data/BSA/wgs/bam_files/ # Change this for different generations
+INPUTPATH=assays/BSA_WGS/dataset #
+OUTPUTPATH=runs/BSA/wgs/bam_files/ # Change this for different generations
mkdir -p $OUTPUTPATH
mkdir -p ${OUTPUTPATH}/metrics/
diff --git a/workflows/BSA/betalain_quantification.Rmd b/workflows/BSA/betalain_quantification.Rmd
index 8ab6265..31a9b69 100644
--- a/workflows/BSA/betalain_quantification.Rmd
+++ b/workflows/BSA/betalain_quantification.Rmd
@@ -20,7 +20,7 @@ library(ggbeeswarm)
Create output directory for plots
```{bash}
-mkdir ../../plots/betalain_quantification
+mkdir ../../runs/betalain_quantification
```
@@ -28,7 +28,7 @@ Read in measured data
```{r}
# read in data
-betalain_quantification <- read_excel(path = "../../raw_data/betalain_quantification/Betalain_quantification_summary.xlsx",
+betalain_quantification <- read_excel(path = "../../assays/betalain_photometric_quantification/dataset/Betalain_quantification_summary.xlsx",
sheet = "Photometric_quantification")
# normalize to the same input weight
@@ -219,11 +219,11 @@ p_leaf <- ggplot(data = melted_quant %>% filter(tissue == "leaf")) +
yend = mean_content),
linewidth = 0.9,
color = "black") +
- geom_text(data = leaf_bc_groups,
+ geom_text(data = leaf_bc_groups,
aes(x = as.numeric(factors) - 0.19,
y = 1.15,
- label = groups),
- size=8,
+ label = groups),
+ size=8,
inherit.aes = F,
color = "red3") +
scale_color_manual(values = c( "red3","#F0B327"), labels = c("Betacyanins", "Betaxanthins")) +
@@ -246,8 +246,8 @@ p_leaf
# plot betalain quantification from flower
p_flower <- ggplot(data = melted_quant %>% filter(tissue == "flower")) +
- # geom_boxplot(aes(x = accession,
- # y = content,
+ # geom_boxplot(aes(x = accession,
+ # y = content,
# fill = metabolite),
# color = "black",
# #color = "red4",
@@ -282,18 +282,18 @@ p_flower <- ggplot(data = melted_quant %>% filter(tissue == "flower")) +
yend = mean_content),
linewidth = 0.9,
color = "black") +
- geom_text(data = flower_bc_groups,
+ geom_text(data = flower_bc_groups,
aes(x = as.numeric(factors) - 0.19,
y = 1.15,
- label = groups),
- size=8,
+ label = groups),
+ size=8,
inherit.aes = F,
color = "red3") +
- geom_text(data = flower_bx_groups,
+ geom_text(data = flower_bx_groups,
aes(x = as.numeric(factors) + 0.19,
y = 1.15,
- label = groups),
- size=8,
+ label = groups),
+ size=8,
inherit.aes = F,
color = "#F0B327") +
scale_fill_manual(values = c( "red3","#F0B327"), labels = c("Betacyanins", "Betaxanthins")) +
@@ -322,7 +322,7 @@ patchplot_betalains <- p_flower / p_leaf +
patchplot_betalains
-ggsave(filename = "../../plots/betalain_quantification/betalain_quant_photometric_content.png",
+ggsave(filename = "../../runs/betalain_quantification/betalain_quant_photometric_content.png",
plot = patchplot_betalains,
bg = "white",
dpi = 450,
@@ -335,7 +335,7 @@ HPLC quantification
```{r}
# hplc quantification
-hplc_quantification <- read_excel(path = "../../raw_data/betalain_quantification/Betalain_quantification_summary.xlsx",
+hplc_quantification <- read_excel(path = "../../assays/betalain_photometric_quantification/dataset/Betalain_quantification_summary.xlsx",
sheet = "LC-MS_quantification")
# get blank measurements
@@ -447,14 +447,14 @@ Plot results amaranthin
```{r}
# plot
pa_leaf <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "leaf")) +
- # geom_point(aes(x = accession,
- # y = Amaranthin_ratio,
+ # geom_point(aes(x = accession,
+ # y = Amaranthin_ratio,
# group = uniq_ind),
# color = "red3",
# position = position_dodge(width = 0.75),
# size = 2.8) +
- geom_beeswarm(aes(x = accession,
- y = Amaranthin_ratio,
+ geom_beeswarm(aes(x = accession,
+ y = Amaranthin_ratio,
group = uniq_ind),
color = "red3",
cex = 1.5,
@@ -489,14 +489,14 @@ pa_leaf <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "leaf")) +
pa_flower <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "flower")) +
- # geom_point(aes(x = accession,
- # y = Amaranthin_ratio,
+ # geom_point(aes(x = accession,
+ # y = Amaranthin_ratio,
# group = uniq_ind),
# color = "red3",
# position = position_dodge(width = 0.75),
# size = 2.8) +
- geom_beeswarm(aes(x = accession,
- y = Amaranthin_ratio,
+ geom_beeswarm(aes(x = accession,
+ y = Amaranthin_ratio,
group = uniq_ind),
color = "red3",
cex = 1.5,
@@ -536,8 +536,8 @@ Plot results betanin
```{r}
# plot
pb_leaf <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "leaf")) +
- geom_beeswarm(aes(x = accession,
- y = Betanin_ratio,
+ geom_beeswarm(aes(x = accession,
+ y = Betanin_ratio,
group = uniq_ind),
color = "red3",
cex = 1.5,
@@ -572,8 +572,8 @@ pb_leaf <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "leaf")) +
pb_flower <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "flower")) +
- geom_beeswarm(aes(x = accession,
- y = Betanin_ratio,
+ geom_beeswarm(aes(x = accession,
+ y = Betanin_ratio,
group = uniq_ind),
color = "red3",
cex = 1.5,
@@ -612,8 +612,8 @@ Plot results betalamic acid
```{r}
# plot
pba_leaf <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "leaf")) +
- geom_beeswarm(aes(x = accession,
- y = Betalamic_acid_ratio,
+ geom_beeswarm(aes(x = accession,
+ y = Betalamic_acid_ratio,
group = uniq_ind),
color = "grey40",
cex = 1.5,
@@ -648,8 +648,8 @@ pba_leaf <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "leaf")) +
pba_flower <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "flower")) +
- geom_beeswarm(aes(x = accession,
- y = Betalamic_acid_ratio,
+ geom_beeswarm(aes(x = accession,
+ y = Betalamic_acid_ratio,
group = uniq_ind),
color = "grey40",
cex = 1.5,
@@ -688,8 +688,8 @@ Plot results vulgaxanthin
```{r}
# plot
pv_leaf <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "leaf")) +
- geom_beeswarm(aes(x = accession,
- y = Vulgaxanthin_IV_ratio,
+ geom_beeswarm(aes(x = accession,
+ y = Vulgaxanthin_IV_ratio,
group = uniq_ind),
color = "#F0B327",
method = "swarm",
@@ -718,8 +718,8 @@ pv_leaf <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "leaf")) +
pv_flower <- ggplot(data = hplc_quant_normalised %>% filter(tissue == "flower")) +
- geom_beeswarm(aes(x = accession,
- y = Vulgaxanthin_IV_ratio,
+ geom_beeswarm(aes(x = accession,
+ y = Vulgaxanthin_IV_ratio,
group = uniq_ind),
color = "#F0B327",
method = "swarm",
@@ -762,7 +762,7 @@ overview_patchplot <- p_leaf + p_flower + pa_leaf + pa_flower + pb_leaf + pb_flo
legend.direction = "vertical",
plot.tag = element_text(vjust = 2, size = 17, face = "bold"))
-ggsave(filename = "../../plots/betalain_quantification/overview_betalain_quantification.png",
+ggsave(filename = "../../runs/betalain_quantification/overview_betalain_quantification.png",
plot = overview_patchplot,
bg = "white",
dpi = 450,
@@ -785,7 +785,7 @@ photo_hplc_patchplot <- plot_spacer() / (p_leaf + p_flower) / (pa_leaf + pa_flow
-ggsave(filename = "../../plots/betalain_quantification/quantification_without_picture.png",
+ggsave(filename = "../../runs/betalain_quantification/quantification_without_picture.png",
plot = photo_hplc_patchplot,
bg = "white",
dpi = 450,
@@ -798,7 +798,7 @@ Analyse HPLC results from amaranth roots:
```{r}
# hplc quantification
-root_quant <- read_excel(path = "../../raw_data/betalain_quantification/Betalain_quantification_summary.xlsx",
+root_quant <- read_excel(path = "../../assays/betalain_photometric_quantification/dataset/Betalain_quantification_summary.xlsx",
sheet = "transgenic_roots_LC-MS_quantification")
root_quant <- root_quant %>%
mutate(sample_id = paste0("plate_", plate, "_", root_type)) %>%
@@ -834,7 +834,7 @@ root_quant_plot <- ggplot(data = root_quant_norm) +
legend.position = "none")
root_quant_plot
-ggsave(filename = "../../plots/betalain_quantification/root_quantification.png",
+ggsave(filename = "../../runs/betalain_quantification/root_quantification.png",
plot = root_quant_plot,
bg = "white",
dpi = 450,
@@ -842,5 +842,3 @@ ggsave(filename = "../../plots/betalain_quantification/root_quantification.png",
height = 5)
```
-
-
diff --git a/workflows/BSA/bsa_and_plotting.R b/workflows/BSA/bsa_and_plotting.R
index 4f9f266..ed0b831 100644
--- a/workflows/BSA/bsa_and_plotting.R
+++ b/workflows/BSA/bsa_and_plotting.R
@@ -1,5 +1,5 @@
-setwd("/home/tom/Documents/projects/Ahyp_v2_2_publication/")
+setwd("/home/tom/Documents/ARC_projects/betalain_regulation_amaranth/")
library(QTLseqr)
library(tidyverse)
@@ -16,7 +16,7 @@ bsa_analysis <- function(rawData,HighBulk,LowBulk,Chroms,nhigh,nlow){
highBulk = HighBulk,
lowBulk = LowBulk,
chromList = Chroms)
-
+
df <- df %>% select(CHROM,
POS,
REF,
@@ -38,7 +38,7 @@ bsa_analysis <- function(rawData,HighBulk,LowBulk,Chroms,nhigh,nlow){
mutate(CHROM=as.factor(as.numeric(gsub("Scaffold_","",CHROM))),
POS=as.numeric(POS)) %>%
filter(REF!='*',ALT!='*')
-
+
df_filt <-filterSNPs(SNPset = df,
refAlleleFreq = 0.2,
minTotalDepth = 50,
@@ -46,7 +46,7 @@ bsa_analysis <- function(rawData,HighBulk,LowBulk,Chroms,nhigh,nlow){
minSampleDepth = 20,
minGQ = 99,
verbose = TRUE)
-
+
df_filt <- runGprimeAnalysis(
SNPset = df_filt,
windowSize = 2e6,
@@ -64,14 +64,14 @@ bsa_analysis <- function(rawData,HighBulk,LowBulk,Chroms,nhigh,nlow){
return(df_filt)
}
-AM_00332_leaf_green_red <- bsa_analysis(rawData = 'data/BSA/wgs/vcf/bulk_snps05.table',
+AM_00332_leaf_green_red <- bsa_analysis(rawData = 'runs/BSA/wgs/vcf/bulk_snps05.table',
HighBulk = "AM_00332_gl",
LowBulk = "AM_00332_rl",
Chroms = paste0(rep("Scaffold_",
16),1:16),
nhigh=80,
nlow=80)
-AM_00331_flower_red_green <- bsa_analysis(rawData = 'data/BSA/wgs/vcf/bulk_snps05.table',
+AM_00331_flower_red_green <- bsa_analysis(rawData = 'runs/BSA/wgs/vcf/bulk_snps05.table',
HighBulk = "AM_00331_rf",
LowBulk = "AM_00331_gf",
Chroms = paste0(rep("Scaffold_",
@@ -83,15 +83,15 @@ AM_00331_flower_red_green <- bsa_analysis(rawData = 'data/BSA/wgs/vcf/bulk_snps0
# plot all results
# leaf
plotGresults <- function(Gresults,betalain_genes){
- qval <- Gresults %>%
- filter(qvalue<=0.01)
+ qval <- Gresults %>%
+ filter(qvalue<=0.01)
#qval <- min(qval$Gprime)
qval <- 3
-
-
+
+
mG <- Gresults %>%
filter(Gprime==max(Gresults$Gprime))
-
+
p1 <- ggplot()+
geom_line(data=Gresults,aes(POS/1e6,Gprime), size=2) +
labs(x= 'Position (Mb)',y= "G' value") +
@@ -99,7 +99,7 @@ plotGresults <- function(Gresults,betalain_genes){
geom_hline(data=data.frame(yint=qval),
aes(yintercept =yint,
linetype ='dashed',
- color=alpha('red',0.6)),
+ color=alpha('red',0.6)),
size=1.7)+
facet_grid(.~CHROM, space = 'free_x',scales='free_x') +
theme(panel.spacing.x=unit(0.25, "lines")) +
@@ -117,23 +117,23 @@ plotGresults <- function(Gresults,betalain_genes){
axis.ticks = element_line(linewidth = 1.5),
axis.ticks.length = unit(.25, "cm")) +
scale_x_continuous(guide = guide_axis(check.overlap = T))
- #geom_gene_arrow(data=betalain_genes,
+ #geom_gene_arrow(data=betalain_genes,
# aes(xmin = start/1e6, xmax = end/1e6, y = max(Gresults$Gprime), fill = type))
-
+
#ggsave(outfile,p1,width = 18,height = 7,,bg='white')
return(p1)
}
# flower
plotGresults1 <- function(Gresults,betalain_genes){
- qval <- Gresults %>%
- filter(qvalue<=0.01)
+ qval <- Gresults %>%
+ filter(qvalue<=0.01)
#qval <- min(qval$Gprime)
qval <- 3
-
-
+
+
mG <- Gresults %>%
filter(Gprime==max(Gresults$Gprime))
-
+
p1 <- ggplot()+
geom_line(data=Gresults,aes(POS/1e6,Gprime), size=2) +
labs(x= 'Position (Mb)',y= "G' value") +
@@ -142,7 +142,7 @@ plotGresults1 <- function(Gresults,betalain_genes){
geom_hline(data=data.frame(yint=qval),
aes(yintercept =yint,
linetype ='dashed',
- color=alpha('red',0.6)),
+ color=alpha('red',0.6)),
size=1.7)+
facet_grid(.~CHROM,space = 'free_x',scales='free_x') +
theme(panel.spacing.x=unit(0.25, "lines")) +
@@ -158,9 +158,9 @@ plotGresults1 <- function(Gresults,betalain_genes){
axis.ticks = element_line(linewidth = 1.5),
axis.ticks.length = unit(.25, "cm")) +
scale_x_continuous(guide = guide_axis(check.overlap = T))
- #geom_gene_arrow(data=betalain_genes,
+ #geom_gene_arrow(data=betalain_genes,
# aes(xmin = start/1e6, xmax = end/1e6, y = max(Gresults$Gprime), fill = type))
-
+
#ggsave(outfile,p1,width = 18,height = 7,,bg='white')
return(p1)
}
@@ -168,23 +168,23 @@ plotGresults1 <- function(Gresults,betalain_genes){
# plot individual chromosomes
# flower
plotGqtl <- function(Gresults,chr,genes){
-
- qval <- Gresults %>%
- filter(qvalue<=0.01)
+
+ qval <- Gresults %>%
+ filter(qvalue<=0.01)
#qval <- min(qval$Gprime)
qval <- 3
my_qtl <- getQTLTable(SNPset = Gresults, alpha = 0.01,export = F)
-
+
ggplot()+
geom_line(data=filter(Gresults,CHROM==chr),aes(POS/1e6,Gprime),size=2) +
labs(x= 'Position (Mb)',y= "G' value") +
scale_x_continuous(breaks = c(0,10,20,30))+
geom_hline(data=data.frame(yint=qval),
- aes(yintercept = yint,
- linetype ='dashed',
+ aes(yintercept = yint,
+ linetype ='dashed',
color=alpha('red',0.6)),
size = 2) +
- facet_grid(.~CHROM,space = 'free_x',scales='free_x') +
+ facet_grid(.~CHROM,space = 'free_x',scales='free_x') +
theme(panel.spacing.x=unit(0.25, "lines")) +
ylim(0,10) +
theme( strip.background = element_rect(fill = alpha('lightblue',0.2)),
@@ -199,19 +199,19 @@ plotGqtl <- function(Gresults,chr,genes){
axis.line = element_line(linewidth = 2),
axis.ticks = element_line(linewidth = 1.5),
axis.ticks.length = unit(.25, "cm")) +
- geom_gene_arrow(data=filter(genes, CHROM==chr, type == "transcript") %>% droplevels(),
+ geom_gene_arrow(data=filter(genes, CHROM==chr, type == "transcript") %>% droplevels(),
aes(xmin = start/1e6, xmax = end/1e6, y = 9.5, color = attributes), size=6) +
scale_color_manual(values = c(alpha('red',0.6), "black", "black","grey","grey"))
}
# leaf
plotGqtl1 <- function(Gresults,chr,genes){
-
- qval <- Gresults %>%
- filter(qvalue<=0.01)
+
+ qval <- Gresults %>%
+ filter(qvalue<=0.01)
#qval <- min(qval$Gprime)
qval <- 3
my_qtl <- getQTLTable(SNPset = Gresults, alpha = 0.01,export = F)
-
+
p1 <- ggplot() +
geom_line(data=filter(Gresults,CHROM==chr),aes(POS/1e6,Gprime),size=2) +
labs(x= 'Position (Mb)',y= "G' value") +
@@ -239,22 +239,22 @@ plotGqtl1 <- function(Gresults,chr,genes){
axis.line = element_line(linewidth = 2),
axis.ticks = element_line(linewidth = 1.5),
axis.ticks.length = unit(.25, "cm"))
-
-
+
+
p2 <- ggplot() +
- geom_gene_arrow(data=filter(genes,
- CHROM==chr,
+ geom_gene_arrow(data=filter(genes,
+ CHROM==chr,
type == "transcript",
attributes == "ID=AHp023147.1;geneID=AHp023147" | attributes == "ID=AHp023148.1;geneID=AHp023148") %>% droplevels(),
- aes(xmin = start,
- xmax = end,
- y = "chr16",
- fill = attributes,
+ aes(xmin = start,
+ xmax = end,
+ y = "chr16",
+ fill = attributes,
forward = c(F,T)),
size = 1.5,
color = "black",
- arrowhead_height = unit(12, "mm"),
- arrowhead_width = unit(6, "mm"),
+ arrowhead_height = unit(12, "mm"),
+ arrowhead_width = unit(6, "mm"),
arrow_body_height = grid::unit(6, "mm")) +
geom_text(aes(x = c(5246000,5290000),
y = "chr16",
@@ -268,7 +268,7 @@ plotGqtl1 <- function(Gresults,chr,genes){
axis.line = element_line(linewidth = 2),
axis.ticks = element_line(linewidth = 1.5),
axis.text.x = element_text(size=20),
- panel.grid.major.y = ggplot2::element_line(colour = "grey",
+ panel.grid.major.y = ggplot2::element_line(colour = "grey",
linewidth = 1),
#axis.title.x = element_text(size=40),
axis.ticks.length = unit(.25, "cm"),
@@ -278,12 +278,12 @@ plotGqtl1 <- function(Gresults,chr,genes){
axis.ticks.y = element_blank(),
axis.text.y = element_blank()) +
scale_fill_manual(values = c("chocolate2","cyan3",'red'))
-
+
# combine plots:
out <- plot_grid(p2,p1,
nrow = 2,
rel_heights = c(0.3,0.7))
-
+
out
}
@@ -348,15 +348,9 @@ MYB_plot <- plot_grid(cowplot_flower, pathway_plot, alignment_plot,
labels = c("", "C", "D"),
label_size = 34)
-ggsave(filename = "plots/paper_myb_combined_alignment.png",
+ggsave(filename = "runs/plots/paper_myb_combined_alignment.png",
plot = MYB_plot,
dpi = 400,
width = 25,
height = 25,
bg = "white")
-
-
-
-
-
-
diff --git a/workflows/BSA/read_count_analysis.Rmd b/workflows/BSA/read_count_analysis.Rmd
index 4154c86..f7c97bb 100644
--- a/workflows/BSA/read_count_analysis.Rmd
+++ b/workflows/BSA/read_count_analysis.Rmd
@@ -11,14 +11,14 @@ library(tidyverse)
library(DESeq2)
library(factoextra)
library(patchwork)
-knitr::opts_knit$set(root.dir = "/home/tom/Documents/projects/Ahyp_v2_2_publication/")
+knitr::opts_knit$set(root.dir = "/home/tom/Documents/ARC_projects/betalain_regulation_amaranth/")
```
```{r}
########################## Create a function to generate plots for all betalain pathway genes
# load object with names of all betalain and flavonoid genes
-pathway_genes <- read.csv(file = "data/manual_sheets/color_pathway_genes.csv", header=T)
+pathway_genes <- read.csv(file = "studies/additional_data/resources/color_pathway_genes.csv", header=T)
colnames(pathway_genes) <- c("pathway_gene", "pathway", "gene_id")
betalain.genes <- pathway_genes %>%
filter(pathway == "Betalain")
@@ -32,13 +32,13 @@ Transcript level gene expression quantification from kallisto:
```{r}
# vector of input directories
-sample_names <- dir(path = "data/flower_color_mapping/kallisto_quant/")
+sample_names <- dir(path = "runs/flower_color_mapping/kallisto_quant/")
# read in tables
kallisto_quant <- c()
for (i in 1:length(sample_names)){
- x <- read_table(file = paste0("data/flower_color_mapping/kallisto_quant/",
+ x <- read_table(file = paste0("runs/flower_color_mapping/kallisto_quant/",
sample_names[i],
"/abundance.tsv"))
# set column names and keep relevant columns
@@ -126,7 +126,7 @@ patchplot <- betalain_plots[[8]] + betalain_plots[[9]] + betalain_plots[[11]] +
theme(plot.margin = unit(c(0.5,0.5,0.5,0.5), "cm"),
plot.tag = element_text(size = 35))
-ggsave(filename = "plots/flower_mapping_expression/betalain_gene_kallisto.png",
+ggsave(filename = "runs/plots/flower_mapping_expression/betalain_gene_kallisto.png",
width = 28,
height = 20)
@@ -182,7 +182,7 @@ betalain_plots <- plot_betalain_counts(gene_ID_list = betalain_quant$transcript_
# save all plots
for (i in 1:length(betalain_plots)){
- ggsave(filename = paste0("plots/flower_mapping_expression/", gene_names[i], ".png"),
+ ggsave(filename = paste0("runs/plots/flower_mapping_expression/", gene_names[i], ".png"),
plot = betalain_plots[[i]],
height = 6,
width = 8)
@@ -219,7 +219,7 @@ ggplot(data=regulator_quant) +
legend.position = "bottom",
legend.direction = "vertical")
-ggsave(filename = "plots/flower_mapping_expression/flower_expression_grid.png",
+ggsave(filename = "runs/plots/flower_mapping_expression/flower_expression_grid.png",
width = 4,
height = 8,
dpi = 500)
@@ -289,7 +289,7 @@ ggplot(data=flavonoid_grid) +
legend.position = "right",
legend.direction = "vertical")
-ggsave(filename = "plots/flower_mapping_expression/flavonoid_expression_grid.png",
+ggsave(filename = "runs/plots/flower_mapping_expression/flavonoid_expression_grid.png",
width = 8,
height = 8,
dpi = 500)
diff --git a/workflows/BSA/read_count_analysis_from_bam.Rmd b/workflows/BSA/read_count_analysis_from_bam.Rmd
index a3d4423..f442e22 100644
--- a/workflows/BSA/read_count_analysis_from_bam.Rmd
+++ b/workflows/BSA/read_count_analysis_from_bam.Rmd
@@ -11,40 +11,40 @@ library(tidyverse)
library(chromstaR)
library(ggtranscript)
library(RColorBrewer)
-knitr::opts_knit$set(root.dir = "/home/tom/Documents/projects/Ahyp_v2_2/")
+knitr::opts_knit$set(root.dir = "/home/tom/Documents/ARC_projects/betalain_regulation_amaranth/")
```
Extract the sequencing reads from the bam files which overlap both the two non-synonymous SNPs and the stop-gained SNP. Samtools view can be used to extract reads covering a particular position (and their pairs). Non-primary alignments can be discarded. Reads should cover the position of the stop-gained variant (Scaffold 16, 5305851, C->T) and the position of the right non-synonymous variant (Scaffold 16, 5305727, A->T).
```{bash}
-mkdir -p data/BSA/RNAseq/phased_reads/
+mkdir -p runs/BSA/RNAseq/phased_reads/
# green flower
# index and extract everything overlapping the right non-synonymous variant position
-samtools index data/BSA/RNAseq/STAR_flower_mappings/AM_00332_gf_Aligned.sortedByCoord.out.bam
-samtools view -b -h -F 256 -P data/BSA/RNAseq/STAR_flower_mappings/AM_00332_gf_Aligned.sortedByCoord.out.bam Scaffold_16:5305727-5305727 > data/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_mismatch.bam
+samtools index runs/BSA/RNAseq/STAR_flower_mappings/AM_00332_gf_Aligned.sortedByCoord.out.bam
+samtools view -b -h -F 256 -P runs/BSA/RNAseq/STAR_flower_mappings/AM_00332_gf_Aligned.sortedByCoord.out.bam Scaffold_16:5305727-5305727 > runs/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_mismatch.bam
# index and extract everything overlapping the stop-gained variant position
-samtools index data/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_mismatch.bam
-samtools view -b -h -P data/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_mismatch.bam Scaffold_16:5305851-5305851 > data/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam
-samtools index data/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam
+samtools index runs/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_mismatch.bam
+samtools view -b -h -P runs/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_mismatch.bam Scaffold_16:5305851-5305851 > runs/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam
+samtools index runs/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam
# save as tsv file using sam2tsv from jvarkit
-java -jar /home/tom/Documents/tools/jvarkit/dist/sam2tsv.jar -R polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta data/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam > data/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam.tsv
+java -jar /home/tom/Documents/tools/jvarkit/dist/sam2tsv.jar -R runs/polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta runs/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam > runs/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam.tsv
# red flower
# index and extract everything overlapping the right non-synonymous variant position
-samtools index data/BSA/RNAseq/STAR_flower_mappings/AM_00332_rf_Aligned.sortedByCoord.out.bam
-samtools view -b -h -F 256 -P data/BSA/RNAseq/STAR_flower_mappings/AM_00332_rf_Aligned.sortedByCoord.out.bam Scaffold_16:5305727-5305727 > data/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_mismatch.bam
+samtools index runs/BSA/RNAseq/STAR_flower_mappings/AM_00332_rf_Aligned.sortedByCoord.out.bam
+samtools view -b -h -F 256 -P runs/BSA/RNAseq/STAR_flower_mappings/AM_00332_rf_Aligned.sortedByCoord.out.bam Scaffold_16:5305727-5305727 > runs/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_mismatch.bam
# index and extract everything overlapping the stop-gained variant position
-samtools index data/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_mismatch.bam
-samtools view -b -h -P data/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_mismatch.bam Scaffold_16:5305851-5305851 > data/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam
-samtools index data/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam
+samtools index runs/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_mismatch.bam
+samtools view -b -h -P runs/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_mismatch.bam Scaffold_16:5305851-5305851 > runs/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam
+samtools index runs/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam
# save as tsv file using sam2tsv from jvarkit
-java -jar /home/tom/Documents/tools/jvarkit/dist/sam2tsv.jar -R polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta data/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam > data/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam.tsv
+java -jar /home/tom/Documents/tools/jvarkit/dist/sam2tsv.jar -R runs/polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta runs/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam > runs/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam.tsv
```
Put in loading and filtering of the data (tsv and sequencing data) into separate functions:
@@ -72,9 +72,9 @@ read_bam.tsv <- function(filename){
arrange(read_name) %>%
ungroup() %>%
#mutate(allele = ifelse(tolower(read_base) == ref_base, "ref", "alt")) %>% # is it the reference or alternative allele?
- mutate(allele = ifelse(ref_pos == 5305722 & read_base == "A",
- "alt",
- ifelse(ref_pos == 5305727 & read_base == "T",
+ mutate(allele = ifelse(ref_pos == 5305722 & read_base == "A",
+ "alt",
+ ifelse(ref_pos == 5305727 & read_base == "T",
"alt",
ifelse(ref_pos == 5305851 & read_base == "T",
"alt", "ref"))))
@@ -104,22 +104,22 @@ plot_reads <- function(df, tsv, title){
# maybe rather join the two tables?
number_alt_alleles <- tsv %>%
filter(allele == "alt") %>%
- group_by(read_name) %>%
+ group_by(read_name) %>%
summarise(n = n(),
snp_grouped = sum(snp_group))
# join the table detailing the number of alt alleles
snp.bam.df <- left_join(df, number_alt_alleles, by = c("qname" = "read_name"))
-
+
# first plot, showing specific reads
snpplot <- ggplot() +
- geom_range(data = snp.bam.df,
+ geom_range(data = snp.bam.df,
aes(xstart = start, xend = end, y = factor(qname, levels=unique(qname[order(n,snp_grouped,qname)]), ordered=TRUE)))
-
+
# second plot showing alternativ allele positions
snpplot2 <- snpplot +
- geom_point(data = tsv %>% filter(allele == "alt"),
- aes(x = ref_pos,
- y = read_name,
+ geom_point(data = tsv %>% filter(allele == "alt"),
+ aes(x = ref_pos,
+ y = read_name,
color = as.factor(snp_group)),
size = 2.2) +
theme_classic() +
@@ -149,9 +149,9 @@ Load in data for the red and green flower bulks of the BSA on AM_00332:
```{r}
# green flower
-snp_tsv.gf <- read_bam.tsv(filename = "data/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam.tsv")
+snp_tsv.gf <- read_bam.tsv(filename = "runs/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam.tsv")
-snp_df.gf <- read_bam_snps_as_df(filename = "data/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam",
+snp_df.gf <- read_bam_snps_as_df(filename = "runs/BSA/RNAseq/phased_reads/AM_00332_gf_Aligned.sortedByCoord.out.covering_both.bam",
tsv = snp_tsv.gf)
# how many reads after filtering?
@@ -159,9 +159,9 @@ snp_tsv.gf %>%
dplyr::count(read_name) # 99 reads
# red flower
-snp_tsv.rf <- read_bam.tsv(filename = "data/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam.tsv")
+snp_tsv.rf <- read_bam.tsv(filename = "runs/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam.tsv")
-snp_df.rf <- read_bam_snps_as_df(filename = "data/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam",
+snp_df.rf <- read_bam_snps_as_df(filename = "runs/BSA/RNAseq/phased_reads/AM_00332_rf_Aligned.sortedByCoord.out.covering_both.bam",
tsv = snp_tsv.rf)
# how many reads after filtering?
@@ -179,7 +179,7 @@ plot.gf <- plot_reads(df = snp_df.gf,
plot.gf
# save plot
-ggsave(filename = "plots/rna_seq_reads_gf.png",
+ggsave(filename = "runs/plots/rna_seq_reads_gf.png",
plot = plot.gf,
width = 10,
height = 8)
@@ -192,17 +192,8 @@ plot.rf <- plot_reads(df = snp_df.rf,
plot.rf
# save plot
-ggsave(filename = "plots/rna_seq_reads_rf.png",
+ggsave(filename = "runs/plots/rna_seq_reads_rf.png",
plot = plot.rf,
width = 10,
height = 8)
```
-
-
-
-
-
-
-
-
-
diff --git a/workflows/BSA/snpEff_analysis.Rmd b/workflows/BSA/snpEff_analysis.Rmd
index f74a6b0..28c1f06 100644
--- a/workflows/BSA/snpEff_analysis.Rmd
+++ b/workflows/BSA/snpEff_analysis.Rmd
@@ -14,7 +14,7 @@ library(ggtranscript)
library(reshape2)
library(cowplot)
library(patchwork)
-knitr::opts_knit$set(root.dir = "/home/tom/Documents/projects/Ahyp_v2_2_publication/")
+knitr::opts_knit$set(root.dir = "/home/tom/Documents/ARC_projects/betalain_regulation_amaranth/")
```
## Database creation
@@ -22,15 +22,15 @@ knitr::opts_knit$set(root.dir = "/home/tom/Documents/projects/Ahyp_v2_2_publicat
Run snpEff database creation on the fixed annotation files. Copy the fixed files to the snpeff directory:
```{bash}
-mkdir -p data/annotation_analysis/snpEff/databases/AHv2.2/
+mkdir -p runs/annotation_analysis/snpEff/databases/AHv2.2/
# snpEff analysis
# add genome file to snpEff database
-cp polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta data/annotation_analysis/snpEff/databases/AHv2.2/sequences.fa
+cp runs/polished_genome_annotation/assembly/Ahypochondriacus_2.2_polished.softmasked.fasta runs/annotation_analysis/snpEff/databases/AHv2.2/sequences.fa
# add annotation file to snpEff database
-cp data/reannotation_correction/manual/Ahypochondriacus_2.2_polished_corrected.gff data/annotation_analysis/snpEff/databases/AHv2.2/genes.gff
-cp data/reannotation_correction/manual/Ahypochondriacus_2.2_polished_corrected.cds.fasta data/annotation_analysis/snpEff/databases/AHv2.2/cds.fa
-cp data/reannotation_correction/manual/Ahypochondriacus_2.2_polished_corrected.prot.fasta data/annotation_analysis/snpEff/databases/AHv2.2/protein.fa
+cp runs/reannotation_correction/manual/Ahypochondriacus_2.2_polished_corrected.gff runs/annotation_analysis/snpEff/databases/AHv2.2/genes.gff
+cp runs/reannotation_correction/manual/Ahypochondriacus_2.2_polished_corrected.cds.fasta runs/annotation_analysis/snpEff/databases/AHv2.2/cds.fa
+cp runs/reannotation_correction/manual/Ahypochondriacus_2.2_polished_corrected.prot.fasta runs/annotation_analysis/snpEff/databases/AHv2.2/protein.fa
# create database:
java -jar /home/tom/Documents/tools/snpEff/snpEff.jar build -v AHv2.2
@@ -42,18 +42,18 @@ java -jar /home/tom/Documents/tools/snpEff/snpEff.jar build -v AHv2.2
Run using data from Markus color/sterility mapping bulks:
```{bash}
-mkdir -p data/annotation_analysis/snpEff/bsa_sterility_color/analysis
+mkdir -p runs/annotation_analysis/snpEff/bsa_sterility_color/analysis
# get data and run snpEff on the example data:
-java -jar /home/tom/Documents/tools/snpEff/snpEff.jar -csvStats data/annotation_analysis/snpEff/bsa_sterility_color/output.stats.csv -v AHv2.2 data/annotation_analysis/snpEff/bsa_sterility_color/gatk_filter_maxmissing05_biallelic.vcf.gz > data/annotation_analysis/snpEff/bsa_sterility_color/output.snpeff.vcf
+java -jar /home/tom/Documents/tools/snpEff/snpEff.jar -csvStats runs/annotation_analysis/snpEff/bsa_sterility_color/output.stats.csv -v AHv2.2 runs/annotation_analysis/snpEff/bsa_sterility_color/gatk_filter_maxmissing05_biallelic.vcf.gz > runs/annotation_analysis/snpEff/bsa_sterility_color/output.snpeff.vcf
# two files are not saved in the output directory but in the current working directory
-mv snpEff_* data/annotation_analysis/snpEff/bsa_sterility_color/
+mv snpEff_* runs/annotation_analysis/snpEff/bsa_sterility_color/
# it is challenging to process the snpEff output for downstream analysis
# snpsift is a software package distributed with snpEff that eases processing
-cat data/annotation_analysis/snpEff/bsa_sterility_color/output.snpeff.chr16.vcf | /home/tom/Documents/tools/snpEff/scripts/vcfEffOnePerLine.pl | java -jar /home/tom/Documents/tools/snpEff/SnpSift.jar extractFields - CHROM POS "ANN[*].GENEID" "ANN[*].EFFECT" > data/annotation_analysis/snpEff/bsa_sterility_color/output.snpeff.chr16.snpsift.txt
+cat runs/annotation_analysis/snpEff/bsa_sterility_color/output.snpeff.chr16.vcf | /home/tom/Documents/tools/snpEff/scripts/vcfEffOnePerLine.pl | java -jar /home/tom/Documents/tools/snpEff/SnpSift.jar extractFields - CHROM POS "ANN[*].GENEID" "ANN[*].EFFECT" > runs/annotation_analysis/snpEff/bsa_sterility_color/output.snpeff.chr16.snpsift.txt
```
@@ -63,11 +63,11 @@ Analyze the snpEff test run and check for high impact variants that can be manua
```{r}
# load in snpEff summary file
-snpEff.tab <- read.table("data/annotation_analysis/snpEff/bsa_sterility_color/snpEff_genes.txt", skip = 1, header = T, comment.char = "")
+snpEff.tab <- read.table("runs/annotation_analysis/snpEff/bsa_sterility_color/snpEff_genes.txt", skip = 1, header = T, comment.char = "")
colnames(snpEff.tab)[1] <- "GeneName"
# load in list of betalain and flavonoid pathway genes
-color_pathways <- read.csv("data/manual_sheets/color_pathway_genes.csv", header = T)
+color_pathways <- read.csv("runs/manual_sheets/color_pathway_genes.csv", header = T)
snpEff.tab <- left_join(snpEff.tab, color_pathways, by = c("GeneId" = "Gene_id"))
# check color pathway genes for high impact variants, moderate and in theory also modifier might also be relevant
@@ -88,14 +88,14 @@ Subset a more detailed table of betalain genes and their respective positions in
```{r}
# load in list of betalain and flavonoid pathway genes
-color_pathways <- read.csv("data/manual_sheets/color_pathway_genes.csv", header = T)
+color_pathways <- read.csv("runs/manual_sheets/color_pathway_genes.csv", header = T)
betalain_chr16 <- color_pathways %>%
filter(Gene_id == "AHp022773" | Gene_id == "AHp023148" | Gene_id == "AHp023147")
# add BvMYB1like gene
betalain_chr16[2,] <- c("BvMYB1like", "Betalain", "AHp022773")
-write.table(betalain_chr16,
- file = "data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.txt",
+write.table(betalain_chr16,
+ file = "runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.txt",
quote = F)
# set up function for reading in a gtf file
@@ -123,18 +123,18 @@ read.gtf <- function(file){
}
# read in annotation
-annotation.gtf <- read.gtf("polished_genome_annotation/annotation/Ahypochondriacus_2.2_polished_corrected.gtf")
+annotation.gtf <- read.gtf("runs/polished_genome_annotation/annotation/Ahypochondriacus_2.2_polished_corrected.gtf")
# subset betalain genes
betalain_chr16.gtf <- annotation.gtf %>%
filter(gene_id %in% betalain_chr16$Gene_id)
-saveRDS(betalain_chr16.gtf, file = "data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.gtf")
+saveRDS(betalain_chr16.gtf, file = "runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.gtf")
#################### generate BED file of relevant positions
-betalain_chr16.gtf <- readRDS("data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.gtf")
+betalain_chr16.gtf <- readRDS("runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.gtf")
# snpeff by default uses the 5000 positions before and after a gene
# create a bed file that can be used to subset the vcf file into relevant variants
betalain_chr16.bed <- betalain_chr16.gtf %>%
@@ -145,16 +145,16 @@ betalain_chr16.bed <- betalain_chr16.gtf %>%
select(chrom, chromStart, chromEnd) %>%
unique()
-write_tsv(betalain_chr16.bed, file = "data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.bed")
+write_tsv(betalain_chr16.bed, file = "runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.bed")
```
Subset the vcf file and extract the allele frequencies using vcftools
```{bash}
# to extract the format field
-vcftools --gzvcf data/BSA/wgs/vcf/gatk_filter_maxmissing05_biallelic.vcf.gz --bed data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.bed --indv AM_00331_gf --indv AM_00331_rf --indv AM_00332_gl --indv AM_00332_rl --extract-FORMAT-info AD --out data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16
+vcftools --gzvcf runs/BSA/wgs/vcf/gatk_filter_maxmissing05_biallelic.vcf.gz --bed runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.bed --indv AM_00331_gf --indv AM_00331_rf --indv AM_00332_gl --indv AM_00332_rl --extract-FORMAT-info AD --out runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16
# also subset the vcf file to include only the variants around the betalain genes
-vcftools --gzvcf data/BSA/wgs/vcf/gatk_filter_maxmissing05_biallelic.vcf.gz --bed data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.bed --indv AM_00331_gf --indv AM_00331_rf --indv AM_00332_gl --indv AM_00332_rl --recode --recode-INFO-all --out data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16
+vcftools --gzvcf runs/BSA/wgs/vcf/gatk_filter_maxmissing05_biallelic.vcf.gz --bed runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.bed --indv AM_00331_gf --indv AM_00331_rf --indv AM_00332_gl --indv AM_00332_rl --recode --recode-INFO-all --out runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16
```
@@ -162,10 +162,10 @@ Plot the annotated variants in betalain genes. In general, it could be interesti
```{r}
# read in betalain gene list on chr 16
-betalain_chr16 <- read.table("data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.txt")
+betalain_chr16 <- read.table("runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.txt")
# read in variant count of extracted SNPs
-allele_depth.tab <- read.table(file = "data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.AD.FORMAT", header = T)
+allele_depth.tab <- read.table(file = "runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.AD.FORMAT", header = T)
allele_depth.tab <- allele_depth.tab %>%
mutate(customid = paste0("16_", POS))
# allele depth denotes first the reference allele and then the alternative allele, only those reads which were involved in allele calling
@@ -176,7 +176,7 @@ allele_depth.tab <- separate(data = allele_depth.tab, col = "AM_00332_gl", sep =
allele_depth.tab <- separate(data = allele_depth.tab, col = "AM_00332_rl", sep = ",", into = c("AM00332_rl_ref", "AM00332_rl_alt"))
# read in snpsift output and subset for betalain genes
-snpsift.tab <- read.table("data/annotation_analysis/snpEff/bsa_sterility_color/output.snpeff.chr16.snpsift.txt", header = T)
+snpsift.tab <- read.table("runs/annotation_analysis/snpEff/bsa_sterility_color/output.snpeff.chr16.snpsift.txt", header = T)
snpsift.tab <- snpsift.tab %>%
filter(ANN....GENEID %in% betalain_chr16$Gene_id) %>%
mutate(customid = paste0("16_", POS)) %>% # add customid column to snpsift table to enable merging of the two tables
@@ -184,7 +184,7 @@ snpsift.tab <- snpsift.tab %>%
# read in betalain gene gtf on chr 16
-betalain_chr16.gtf <- readRDS("data/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.gtf")
+betalain_chr16.gtf <- readRDS("runs/annotation_analysis/snpEff/bsa_sterility_color/analysis/betalain_chr16.gtf")
# join the snpsift table with the allele depth table
joined.df <- left_join(snpsift.tab, allele_depth.tab, by = c("CHROM", "POS", "customid"))
@@ -201,7 +201,7 @@ Set up plotting functions:
filter_variants <- function(variants, gene, bulk1_ref, bulk1_alt, bulk2_ref, bulk2_alt){
# prepare data by only keeping the relevant gene variants and samples
- dat <- variants %>%
+ dat <- variants %>%
filter(ANN....GENEID == gene) %>%
select(CHROM, POS, ANN....EFFECT, bulk1_ref, bulk1_alt, bulk2_ref, bulk2_alt) %>%
filter(bulk1_alt != 0 & bulk2_alt != 0)
@@ -226,13 +226,13 @@ plot_bulk_comparison <- function(annotation, trans_id, filtered_variants){
filter(transcript_id == trans_id,
type == "CDS")
filtered_variants <- filtered_variants[filtered_variants$POS >= min(annotation.filtered$start) & filtered_variants$POS <= max(annotation.filtered$end),]
- filtered_variants$ANN....EFFECT <- factor(filtered_variants$ANN....EFFECT, levels = c("intron_variant",
- "synonymous_variant",
+ filtered_variants$ANN....EFFECT <- factor(filtered_variants$ANN....EFFECT, levels = c("intron_variant",
+ "synonymous_variant",
"missense_variant",
"stop_gained"))
min_pos <- min(annotation.filtered$start)
max_pos <- max(annotation.filtered$end)
-
+
# plot the gene with variants
p2 <- ggplot() +
geom_range(data = annotation.filtered,
@@ -253,7 +253,7 @@ plot_bulk_comparison <- function(annotation, trans_id, filtered_variants){
x = "Position Scaffold 16 (bp)") +
theme_classic() +
#scale_fill_brewer(palette = "RdBl", direction = -1) + # think about color palette to use
- scale_fill_viridis_d(direction = -1,
+ scale_fill_viridis_d(direction = -1,
labels = c("Intron variant", "Synonymous variant", "Missense variant", "Stop gained")) +
theme(text = element_text(size = 21),
#legend.position = "none",
@@ -277,23 +277,23 @@ plot_bulk_comparison <- function(annotation, trans_id, filtered_variants){
out_plot <- plot_grid(p2, legend,
nrow = 1,
rel_widths = c(0.75, 0.25))
-
+
# # rearrange data:
# dat1 <- filtered_variants[,-(4:5)]
# dat2 <- filtered_variants[,-(6:7)]
# dat1.melt <- melt(dat1, id.vars = c("CHROM", "POS", "ANN....EFFECT"))
# dat2.melt <- melt(dat2, id.vars = c("CHROM", "POS", "ANN....EFFECT"))
- #
+ #
# # plot relative allele frequency
# p1 <- ggplot() +
# geom_col(data = dat1.melt,
# aes(x = POS, y = as.numeric(value), fill = variable), position = "stack", width = 20) +
# xlim(c(min_pos, max_pos)) +
- # labs(y = "",
- # fill = "red_bulk",
+ # labs(y = "",
+ # fill = "red_bulk",
# x = "") +
# theme_classic() +
- # scale_fill_brewer(palette = "Set1",
+ # scale_fill_brewer(palette = "Set1",
# labels = c("Reference allele", "Alternative allele"),
# direction = -1,
# guide = guide_legend(override.aes = list(alpha = 0))) + # make legend invisible
@@ -307,12 +307,12 @@ plot_bulk_comparison <- function(annotation, trans_id, filtered_variants){
# axis.title.x = element_blank(),
# legend.title = element_text(color = "transparent"),
# legend.text = element_text(color = "transparent"))
- #
+ #
# bars <- map(unique(dat2.melt$POS)
# , ~geom_col(position = "stack",
# width = 20
# , data = dat2.melt %>% filter(POS == .x)))
- #
+ #
# p3 <- ggplot(data = dat2.melt,
# aes(x=POS,
# y=as.numeric(value),
@@ -322,8 +322,8 @@ plot_bulk_comparison <- function(annotation, trans_id, filtered_variants){
# labs(fill = "green_bulk",
# x = "Position on Scaffold 16") +
# theme_classic() +
- # scale_fill_brewer(palette = "Set1",
- # labels = c("Reference allele", "Alternative allele"),
+ # scale_fill_brewer(palette = "Set1",
+ # labels = c("Reference allele", "Alternative allele"),
# direction = -1,
# guide = guide_legend(override.aes = list(alpha = 0))) +
# theme(text = element_text(size = 21),
@@ -331,9 +331,9 @@ plot_bulk_comparison <- function(annotation, trans_id, filtered_variants){
# legend.position = "none",
# legend.title = element_text(color = "transparent"),
# legend.text = element_text(color = "transparent"))
-
-
- #allplots <- p1 + p2 + p3 +
+
+
+ #allplots <- p1 + p2 + p3 +
#plot_layout(ncol = 1)
return(out_plot)
}
@@ -346,7 +346,7 @@ Plot for CYP76AD2:
```{r}
# plot for one gene
# filter all homozygous reference variants
-dat <- joined.df %>%
+dat <- joined.df %>%
filter(ANN....GENEID == "AHp023148") %>%
filter(AM00332_gl_alt != 0)
@@ -355,7 +355,7 @@ dat <- joined.df %>%
# filter to only include specific gene
-dat.filtered <- filter_variants(variants = joined.df,
+dat.filtered <- filter_variants(variants = joined.df,
gene = "AHp023148",
bulk1_ref = "AM00332_gl_ref",
bulk1_alt = "AM00332_gl_alt",
@@ -373,8 +373,8 @@ AM00332_CYP76AD <- plot_bulk_comparison(annotation = betalain_chr16.gtf,
AM00332_CYP76AD
-ggsave(filename = "plots/CYP76AD_AHp023148_bsa_snpeff.png",
- width = 14,
+ggsave(filename = "runs/plots/CYP76AD_AHp023148_bsa_snpeff.png",
+ width = 14,
height = 6)
```
@@ -387,19 +387,19 @@ plot_all_genes <- function(gene_id, transcript_id, bulk){
output <- list()
for (i in 1:length(transcript_id)){
# filter all homozygous reference variants
- dat <- joined.df %>%
+ dat <- joined.df %>%
filter(ANN....GENEID == gene_id[i]) %>%
filter(AM00332_gl_alt != 0)
# filter to only include specific gene
if (bulk == "AM00332"){
- dat.filtered <- filter_variants(variants = joined.df,
+ dat.filtered <- filter_variants(variants = joined.df,
gene = gene_id[i],
bulk1_ref = "AM00332_gl_ref",
bulk1_alt = "AM00332_gl_alt",
bulk2_ref = "AM00332_rl_ref",
bulk2_alt = "AM00332_rl_alt")
} else if (bulk == "AM00331"){
- dat.filtered <- filter_variants(variants = joined.df,
+ dat.filtered <- filter_variants(variants = joined.df,
gene = gene_id[i],
bulk1_ref = "AM00331_gf_ref",
bulk1_alt = "AM00331_gf_alt",
@@ -425,9 +425,9 @@ bulk_AM00332_plot_list <- plot_all_genes(gene_id = gene_id,
bulk = "AM00332")
for (i in 1:length(bulk_AM00332_plot_list)){
- ggsave(filename = paste0("plots/BSA/AM00332_color_loss_", gene_id[i], ".png"),
+ ggsave(filename = paste0("runs/plots/BSA/AM00332_color_loss_", gene_id[i], ".png"),
plot = bulk_AM00332_plot_list[[i]],
- width = 14,
+ width = 14,
height = 6)
}
@@ -437,9 +437,9 @@ bulk_AM00331_plot_list <- plot_all_genes(gene_id = gene_id,
bulk = "AM00331")
for (i in 1:length(bulk_AM00331_plot_list)){
- ggsave(filename = paste0("plots/BSA/AM00331_color_loss_", gene_id[i], ".png"),
+ ggsave(filename = paste0("runs/plots/BSA/AM00331_color_loss_", gene_id[i], ".png"),
plot = bulk_AM00331_plot_list[[i]],
- width = 14,
+ width = 14,
height = 6)
}
```
@@ -456,8 +456,8 @@ plot_bulk_comparison_zoom <- function(annotation, trans_id, filtered_variants){
filter(transcript_id == trans_id,
type == "CDS")
filtered_variants <- filtered_variants[filtered_variants$POS >= min(annotation.filtered$start) & filtered_variants$POS <= max(annotation.filtered$end),]
- filtered_variants$ANN....EFFECT <- factor(filtered_variants$ANN....EFFECT, levels = c("intron_variant",
- "synonymous_variant",
+ filtered_variants$ANN....EFFECT <- factor(filtered_variants$ANN....EFFECT, levels = c("intron_variant",
+ "synonymous_variant",
"missense_variant",
"stop_gained"))
# plot the gene with variants
@@ -486,26 +486,26 @@ plot_bulk_comparison_zoom <- function(annotation, trans_id, filtered_variants){
axis.line.x = element_blank(),
axis.text.x = element_blank(),
legend.position = "none") # this increases the legend margin
- # margin has to be increased so that other legends are not cut off,
+ # margin has to be increased so that other legends are not cut off,
# since the first legend seems to determine the margins
-
+
# rearrange data:
dat1 <- filtered_variants[,-(4:5)]
dat2 <- filtered_variants[,-(6:7)]
dat1.melt <- melt(dat1, id.vars = c("CHROM", "POS", "ANN....EFFECT"))
dat2.melt <- melt(dat2, id.vars = c("CHROM", "POS", "ANN....EFFECT"))
-
+
# plot relative allele frequency
p1 <- ggplot() +
geom_col(data = dat1.melt,
aes(x = POS, y = as.numeric(value), fill = variable), position = "stack", width = 2) +
xlim(c(min(annotation.filtered %>% select(start)),
max(annotation.filtered %>% select(end)))) +
- labs(y = "Red bulk\n allele depth",
- fill = "red_bulk",
+ labs(y = "Red bulk\n allele depth",
+ fill = "red_bulk",
x = "") +
theme_classic() +
- scale_fill_brewer(palette = "Set1",
+ scale_fill_brewer(palette = "Set1",
labels = c("Reference allele", "Alternative allele"),
direction = -1,
guide = guide_legend(override.aes = list(alpha = 0))) + # make legend invisible
@@ -524,12 +524,12 @@ plot_bulk_comparison_zoom <- function(annotation, trans_id, filtered_variants){
axis.title.x = element_blank(),
legend.title = element_text(color = "transparent"),
legend.text = element_text(color = "transparent"))
-
+
bars <- map(unique(dat2.melt$POS)
, ~geom_col(position = "stack",
width = 2
, data = dat2.melt %>% filter(POS == .x)))
-
+
p3 <- ggplot(data = dat2.melt,
aes(x=POS,
y=as.numeric(value),
@@ -541,7 +541,7 @@ plot_bulk_comparison_zoom <- function(annotation, trans_id, filtered_variants){
fill = "",
y = "Green bulk\n allele depth") +
theme_classic() +
- scale_fill_brewer(palette = "Set1",
+ scale_fill_brewer(palette = "Set1",
labels = c("Reference allele", "Alternative allele"),
#guide = guide_legend(override.aes = list(alpha = 0)),
direction = -1) +
@@ -564,13 +564,13 @@ plot_bulk_comparison_zoom <- function(annotation, trans_id, filtered_variants){
p3 <- p3 + theme(legend.position = "none")
# combine the three plots
allplots <- plot_grid(p1, p2, p3,
- ncol = 1,
+ ncol = 1,
align = "v",
rel_heights = c(0.3, 0.25, 0.45))
-
-
+
+
# combine other plots with legend
- allplots <- plot_grid(legend, allplots,
+ allplots <- plot_grid(legend, allplots,
ncol = 1,
#align = "v",
rel_heights = c(0.2,0.8))
@@ -582,7 +582,7 @@ AM00332_CYP76AD_zoom <- plot_bulk_comparison_zoom(annotation = betalain_chr16.gt
filtered_variants = dat.filtered)
AM00332_CYP76AD_zoom
-ggsave(filename = "plots/CYP76AD_AHp023148_bsa_snpeff_zoom.png")
+ggsave(filename = "runs/plots/CYP76AD_AHp023148_bsa_snpeff_zoom.png")
```
@@ -620,16 +620,10 @@ grid_with_BSA <- plot_grid(cowplot_leaf, complete_grid,
#grid_with_BSA
# save plot
-ggsave(filename = "plots/BSA_with_grid.png",
+ggsave(filename = "runs/plots/BSA_with_grid.png",
plot = grid_with_BSA,
width = 25,
height = 20,
bg = "white",
dpi = 500)
```
-
-
-
-
-
-
diff --git a/workflows/genome_polishing/helper_script.R b/workflows/genome_polishing/helper_script.R
index 2a93648..1571dce 100644
--- a/workflows/genome_polishing/helper_script.R
+++ b/workflows/genome_polishing/helper_script.R
@@ -1,20 +1,20 @@
# set working directory
-setwd("/home/tom/Documents/projects/Ahyp_v2_2_publication/")
+setwd("/home/tom/Documents/ARC_projects/betalain_regulation_amaranth/")
# read in list of all headers with the correct order
-headers <- read.table("data/NextPolish/input/out.headers.txt")
+headers <- read.table("runs/NextPolish/input/out.headers.txt")
headers <- gsub(">","",headers$V1)
headers <- gsub("quiver_","quiver",headers)
# read in prefiltered fasta index
-prefilter <- read.table("data/NextPolish/input/out.prefiltered.renamed.txt.fai")
+prefilter <- read.table("runs/NextPolish/input/out.prefiltered.renamed.txt.fai")
-# use the
-# every sequence that is in
+# use the
+# every sequence that is in
no_seq <- headers[!headers %in% prefilter[,1]]
no_seq <- sub("",">",no_seq)
-write.table(no_seq, file="data/NextPolish/processed/header_without_sequence.fa",
+write.table(no_seq, file="runs/NextPolish/processed/header_without_sequence.fa",
quote=F,
row.names = F,
col.names = F)
diff --git a/workflows/genome_polishing/process_nextpolish_output.sh b/workflows/genome_polishing/process_nextpolish_output.sh
index 65ab458..d24e306 100644
--- a/workflows/genome_polishing/process_nextpolish_output.sh
+++ b/workflows/genome_polishing/process_nextpolish_output.sh
@@ -4,8 +4,8 @@
# (see master_thesis/code/process_nextpolish_output.sh for more information about the input file preparation)
# Setup
-NPOUT=data/NextPolish/output/
-NPPROCESSED=data/NextPolish/processed/
+NPOUT=runs/NextPolish/output/
+NPPROCESSED=runs/NextPolish/processed/
mkdir -p "$NPPROCESSED"
@@ -15,10 +15,10 @@ mkdir -p "$NPPROCESSED"
cut -f1,2 -d'_' "$NPOUT"genome.nextpolish.fa > "$NPPROCESSED"genome.nextpolish.renamed.fa
# index the prefiltered fasta file for use in R
-samtools faidx data/NextPolish/input/out.prefiltered.renamed.txt
+samtools faidx runs/NextPolish/input/out.prefiltered.renamed.txt
# filter the prefiltered file for everything that is not in Nextpolish file:
-/home/tom/Documents/tools/bbmap/filterbyname.sh in=data/NextPolish/input/out.prefiltered.renamed.txt \
+/home/tom/Documents/tools/bbmap/filterbyname.sh in=runs/NextPolish/input/out.prefiltered.renamed.txt \
names="$NPPROCESSED"genome.nextpolish.renamed.fa \
out="$NPPROCESSED"prefilter_not_in_Nextpolish.fa
@@ -34,7 +34,7 @@ cat "$NPPROCESSED"genome.nextpolish.renamed.fa \
LC_ALL=C awk -v RS=">" -v FS="\n" -v ORS="\n" -v OFS="" '$0 {$1=">"$1"\n"; print}' "$NPPROCESSED"combined.fa > "$NPPROCESSED"combined.linear.fa
# rename header file by removing trailing underscore character of Contigs:
-sed 's/quiver_/quiver/' data/NextPolish/input/out.headers.txt > "$NPPROCESSED"out.header.renamed.txt
+sed 's/quiver_/quiver/' runs/NextPolish/input/out.headers.txt > "$NPPROCESSED"out.header.renamed.txt
# order file:
ORDER="$NPPROCESSED"out.header.renamed.txt
@@ -60,5 +60,5 @@ rm "$NPPROCESSED"out*
rm "$NPPROCESSED"prefilter*
# copy to final output directory
-mkdir -p polished_reference_genome/polished_genome_annotation/assembly/
-cp "$NPPROCESSED"Ahypochondriacus_2.2_polished.fasta polished_reference_genome/polished_genome_annotation/assembly/
+mkdir -p runs/polished_reference_genome/polished_genome_annotation/assembly/
+cp "$NPPROCESSED"Ahypochondriacus_2.2_polished.fasta runs/polished_reference_genome/polished_genome_annotation/assembly/
diff --git a/workflows/genome_polishing/remove_ambiguous_bases.sh b/workflows/genome_polishing/remove_ambiguous_bases.sh
index c810a8a..1cb8a10 100644
--- a/workflows/genome_polishing/remove_ambiguous_bases.sh
+++ b/workflows/genome_polishing/remove_ambiguous_bases.sh
@@ -6,8 +6,8 @@
# N specific entries can in a last step be removed. To reconstruct, the saved order can be used to integrate the N chromosomes again (which record the number of Ns removed)
# Change these two parameters
-INPUT=reference_genomes/Ahypochondriacus/assembly/Ahypochondriacus_459_v2.0.nospace.underscore.fa
-OUTDIR=data/NextPolish/input/
+INPUT=studies/additional_data/resources/reference_genomes/Ahypochondriacus/assembly/Ahypochondriacus_459_v2.0.nospace.underscore.fa
+OUTDIR=runs/NextPolish/input/
mkdir -p $OUTDIR
@@ -37,9 +37,8 @@ grep ">" "$OUTDIR"tmp2.txt > "$OUTDIR"out.headers.txt
echo "headers saved"
# remove headers without sequence (Can be caused by stretch of Ns at the start of a Scaffold (see Scaffold 10))
-sed -r 'N; /(>)[^\n]*\n\1/ s/[^\n]*//; P; D' "$OUTDIR"tmp2.txt | grep . | grep -i -B 1 --no-group-separator '[ATGC]' > "$OUTDIR"data/NextPolish/input/Ahypochondriacus_split.fasta
+sed -r 'N; /(>)[^\n]*\n\1/ s/[^\n]*//; P; D' "$OUTDIR"tmp2.txt | grep . | grep -i -B 1 --no-group-separator '[ATGC]' > "$OUTDIR"Ahypochondriacus_split.fasta
# remove and rename temporary files
rm "$OUTDIR"tmp.txt
mv "$OUTDIR"tmp2.txt "$OUTDIR"out.prefiltered.txt
-
diff --git a/workflows/genome_polishing/run_nextpolish.sh b/workflows/genome_polishing/run_nextpolish.sh
index cbf0579..a086c0b 100644
--- a/workflows/genome_polishing/run_nextpolish.sh
+++ b/workflows/genome_polishing/run_nextpolish.sh
@@ -19,15 +19,15 @@ module load samtools
mkdir -p /scratch/twinkle1/nextpolish/
# set output directory for saving the polished genome to:
-OUTDIR=data/NextPolish/output/
+OUTDIR=runs/NextPolish/output/
### Prepare input files:
# remove all reads containing ambiguous bases from the input using bbduk
/home/twinkle1/tools/bbmap/bbduk.sh maxns=0 \
- in=/projects/ag-stetter/twinkle/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_1.fastq.gz \
- in2=/projects/ag-stetter/twinkle/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_2.fastq.gz \
+ in=/studies/additional_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_1.fastq.gz \
+ in2=/studies/additional_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_2.fastq.gz \
out=/scratch/twinkle1/SRR2106212_1.cleaned.fq \
out2=/scratch/twinkle1/SRR2106212_2.cleaned.fq \
-Xmx16g
@@ -49,7 +49,7 @@ round=2
threads=20
read1=/scratch/twinkle1/SRR2106212_1.cleaned.repair.fq
read2=/scratch/twinkle1/SRR2106212_2.cleaned.repair.fq
-input=/home/twinkle1/master_thesis/data/NextPolish/input/Ahypochondriacus_split.fasta
+input=/runs/NextPolish/input/Ahypochondriacus_split.fasta
for ((i=1; i<=${round};i++)); do
diff --git a/workflows/genome_polishing/unpackSRA.sh b/workflows/genome_polishing/unpackSRA.sh
index 7467f47..6cdf885 100644
--- a/workflows/genome_polishing/unpackSRA.sh
+++ b/workflows/genome_polishing/unpackSRA.sh
@@ -14,29 +14,29 @@
# download file, show progress, increase default max size so that the download starts
# tools/sratoolkit.2.11.2-centos_linux64/bin/prefetch -p -O /projects/ag-stetter/twinkle/lightfoot_WGS_short_reads/ --max-size 30G SRR2106212
-QCOUT=data/NextPolish/QC
+QCOUT=runs/NextPolish/QC
# set working directory
-cd /projects/ag-stetter/twinkle/projects/Ahyp_v2_2_publication/raw_data/lightfoot_WGS_short_reads/
+cd /studies/additional_data/lightfoot_WGS_short_reads/
# before running the fastq-dump command, switch off "Enable Remote Access" by running sratoolskit/bin/vdb-config -i
# split into fastq files
/home/twinkle1/tools/sratoolkit.2.11.2-centos_linux64/bin/fastq-dump --split-3 --verbose SRR2106212.sra
# set working directory
-cd /projects/ag-stetter/twinkle/projects/Ahyp_v2_2_publication/
+cd /projects/ag-stetter/twinkle/ARC_projects/betalain_regulation_amaranth
# gzip the resulting fastq files
# Even though there is an option to gzip it directly using the fastq-dump command, the option is deprecated and should no longer be used
-gzip raw_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_1.fastq
-gzip raw_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_2.fastq
+gzip /studies/additional_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_1.fastq
+gzip /studies/additional_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_2.fastq
# remove sra file afterwards
-rm raw_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212.sra
+rm /studies/additional_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212.sra
# quality control:
module load fastqc/0.11.9
-fastqc -o raw_data/lightfoot_WGS_short_reads/QC/ -t 8 \
- raw_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_1.fastq.gz \
- raw_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_2.fastq.gz
+fastqc -o /studies/additional_data/lightfoot_WGS_short_reads/QC/ -t 8 \
+ /studies/additional_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_1.fastq.gz \
+ /studies/additional_data/lightfoot_WGS_short_reads/SRR2106212/SRR2106212_2.fastq.gz
--
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