An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae V.4

DATA="path_to_zipped_fastq_files"
SAMPLES="file_listing_fastq_files_to_process"
DIRECTORY="path_to_outfiles"
BBMAP_RESOURCES="path_to_bbmap_reference_files"
GENOME_scer="reference_S.cerevisiae_genome_file"
BED_scer="reference_S.cerevisiae_bed_file"

mkdir -p $DIRECTORY/logs/fastqc/fastq
mkdir -p $DIRECTORY/logs/multiqc/fastq
mkdir -p $DIRECTORY/fastp
mkdir -p $DIRECTORY/logs/fastp
mkdir -p $DIRECTORY/bbduk
mkdir -p $DIRECTORY/logs/bbduk
mkdir -p $DIRECTORY/logs/fastqc/bbduk
mkdir -p $DIRECTORY/logs/multiqc/bbduk
mkdir -p $DIRECTORY/slamdunk_scer
mkdir -p $DIRECTORY/slamdunk_scer/count_twotcreadcount
mkdir -p $DIRECTORY/slamdunk_scer/alleyoop
mkdir -p $DIRECTORY/logs/multiqc/slamdunk_scer

echo "running fastqc on raw data for sample:"
for sample_name in $(cat $SAMPLES | sed "s/_001.fastq.gz//" | uniq)
do
	echo "  ${sample_name}..."

	(fastqc \
		$DATA/${sample_name}_001.fastq.gz \
		--outdir $DIRECTORY/logs/fastqc/fastq \
		--threads 64 \
		--quiet \
		&> /dev/null)

done


echo "running multiqc on raw data..."
multiqc	\
	$DIRECTORY/logs/fastqc/fastq \
	--outdir $DIRECTORY/logs/multiqc/fastq \
	--quiet \
	&> /dev/null

echo "processing UMIs for sample:"
for sample_name in $(cat $SAMPLES | sed "s/_R._001.fastq.gz//" | uniq)
do
	echo "  ${sample_name}..."

	(fastp \
		--in1 $DATA/${sample_name}_R1_001.fastq.gz \
		--out1 $DIRECTORY/fastp/${sample_name}_umi_R1_001.fastq.gz \
		--in2 $DATA/${sample_name}_R2_001.fastq.gz \
		--out2 $DIRECTORY/fastp/${sample_name}_umi_R2_001.fastq.gz \
		--disable_adapter_trimming \
		--disable_quality_filtering \
		--disable_length_filtering \
		--umi \
		--umi_loc read2 \
		--umi_len 12 \
		--json $DIRECTORY/logs/fastp/${sample_name}_umi_fastp.json \
		--html $DIRECTORY/logs/fastp/${sample_name}_umi_fastp.html \
		2> $DIRECTORY/logs/fastp/${sample_name}_umi_fastp.log)

done

echo "trimming adapters for sample:"
for sample_name in $(ls -1 $DIRECTORY/fastp | sed "s/_umi_R._001.fastq.gz//" | uniq)
do
	echo "  ${sample_name}..."

	(bbduk.sh \
		in=$DIRECTORY/fastp/${sample_name}_umi_R1_001.fastq.gz \
		out=$DIRECTORY/bbduk/${sample_name}_trimmed_R1_001.fastq.gz \
		ref=$BBMAP_RESOURCES/truseq.fa.gz,$BBMAP_RESOURCES/polyA.fa.gz \
		k=13 \
		ktrim=r \
		mink=5 \
		qtrim=r \
		trimq=10 \
		minlength=20 \
		threads=16 \
		2> $DIRECTORY/logs/bbduk/${sample_name}_trimmed_bbduk.log)

done

echo "running fastqc on preprocessed data..."
fastqc \
	$DIRECTORY/bbduk/*.fastq.gz \
	--outdir $DIRECTORY/logs/fastqc/bbduk \
	--threads 64 \
	--quiet \
	&> /dev/null


echo "running multiqc on preprocessed data..."
multiqc	\
	$DIRECTORY/logs/fastqc/bbduk \
	--outdir $DIRECTORY/logs/multiqc/bbduk \
	--quiet \
	&> /dev/null

echo "running slamdunk all..."
slamdunk all \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--outputDir $DIRECTORY/slamdunk_scer \
	--trim-5p 5 \
	--topn 100 \
	--multimap \
	--min-coverage 10 \
	--var-fraction 0.8 \
	--max-read-length 151 \
	--min-base-qual 26 \
	--skip-sam \
	--threads 24 \
	$DIRECTORY/bbduk/*.fastq.gz

mv $DIRECTORY/slamdunk_scer/count $DIRECTORY/slamdunk_scer/count_onetcreadcount

echo "running slamdunk count..."
slamdunk count \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--outputDir $DIRECTORY/slamdunk_scer/count_twotcreadcount \
	--max-read-length 151 \
	--min-base-qual 26 \
	--conversion-threshold 2 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam

echo "running alleyoop..."
alleyoop rates \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/rates \
	--reference $GENOME_scer \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop tccontext \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/tccontext \
	--reference $GENOME_scer \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop utrrates \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/utrrates \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--multiTCStringency \
	--max-read-length 151 \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop snpeval \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/snpeval \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--min-coverage 10 \
	--var-fraction 0.8 \
	--multiTCStringency \
	--max-read-length 151 \
	--min-base-qual 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop summary \
	--output $DIRECTORY/slamdunk_scer/alleyoop/summary_onetcreadcount.tsv \
	--tcountDir $DIRECTORY/slamdunk_scer/count_onetcreadcount \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop summary \
	--output $DIRECTORY/slamdunk_scer/alleyoop/summary_twotcreadcount.tsv \
	--tcountDir $DIRECTORY/slamdunk_scer/count_twotcreadcount \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop merge \
	--output $DIRECTORY/slamdunk_scer/alleyoop/merge_totalreadcount.tsv \
	--column "ReadCount" \
	$DIRECTORY/slamdunk_scer/count_onetcreadcount/*.tsv


alleyoop merge \
	--output $DIRECTORY/slamdunk_scer/alleyoop/merge_onetcreadcount.tsv \
	--column "TcReadCount" \
	$DIRECTORY/slamdunk_scer/count_onetcreadcount/*.tsv


alleyoop merge \
	--output $DIRECTORY/slamdunk_scer/alleyoop/merge_twotcreadcount.tsv \
	--column "TcReadCount" \
	$DIRECTORY/slamdunk_scer/count_twotcreadcount/*.tsv


alleyoop tcperreadpos \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/tcperreadpos \
	--reference $GENOME_scer \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--max-read-length 151 \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop tcperutrpos \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/tcperutrpos \
	--reference $GENOME_scer \
	--bed $BED_scer \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--max-read-length 151 \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam


alleyoop dump \
	--outputDir $DIRECTORY/slamdunk_scer/alleyoop/dump \
	--reference $GENOME_scer \
	--snp-directory $DIRECTORY/slamdunk_scer/snp \
	--min-basequality 26 \
	--threads 24 \
	$DIRECTORY/slamdunk_scer/filter/*.bam

echo "running multiqc on slamdunk output..."
multiqc\
	$DIRECTORY/slamdunk_scer/alleyoop \
	--outdir $DIRECTORY/logs/multiqc/slamdunk_scer \
	--quiet \
	&> /dev/null

GENOME_spom="reference_S.pombe_genome_file"
BED_spom="reference_S.pombe_bed_file"

mkdir -p $DIRECTORY/slamdunk_spom
mkdir -p $DIRECTORY/slamdunk_spom/count_conversionthreshold
mkdir -p $DIRECTORY/slamdunk_spom/alleyoop
mkdir -p $DIRECTORY/logs/multiqc/slamdunk_spom