FLASH-seq protocol V.3

# 0. Variables
BASECALL_DIR="/path/to/flowcell/Data/Intensities/BaseCalls/"
OUTPUT_DIR="/path/to/output_folder/"
SAMPLESHEET="/path/to/Demultiplexing_SampleSheet.csv"

# 1. Bcl2fastq
ulimit -n 10000
cd /path/to/flowcell/
bcl2fastq --input-dir $BASECALL_DIR --output-dir $OUTPUT_DIR --sample-sheet $SAMPLESHEET --create-fastq-for-index-reads --no-lane-splitting

zcat Undetermined_S0_I1_001.fastq.gz | awk -F' 1:N:0:' 'NR%4==1{print $2}' | sort | uniq -c > left_index.txt
sort -k1,1 left_index.txt

for file in ./out/*R1*
do
zcat $file | wc -l 
done

# 0. Variables
OUTPUTREF="/path/to/STAR_indexed_genome/"
FASTA="GRCh38.primary_assembly.genome.fa"
GTF="gencode.v34.primary_assembly.annotation.gtf"

# 1. Genome indexing
# sjdbOverhang should be adapted based on the read length (read_length - 1)
mkdir $OUTPUTREF

STAR --runThreadN 15 --runMode genomeGenerate --genomeDir $OUTPUTREF --genomeFastaFiles $FASTA --sjdbGTFfile $GTF --sjdbOverhang 74

bbduk.sh -Xmx48g in=sample.fastq.gz out=cleaned.left.fastq t=32 ktrim=l ref=adapters.fa k=23 mink=7 hdist=1 hdist2=0 tbo
bbduk.sh -Xmx48g in=cleaned.left.fastq out=cleaned.fastq t=32 ktrim=r ref=adapters.fa k=23 mink=7 hdist=1 hdist2=0 tbo

mv FASTQ/cleaned.fastq FASTQ/sample.R1.fastq.gz

# 0. Variables
GENOME="/path/to/STAR_indexed_genome/"
FASTQ="/path/to/sample.R1.fastq.gz"
ID=”sample_id”

# 1. Mapping
STAR --runThreadN 30 --limitBAMsortRAM 20000000000 --genomeLoad LoadAndKeep --genomeDir "$GENOME" --readFilesIn "$FASTQ" --readFilesCommand zcat --limitSjdbInsertNsj 2000000 --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outSAMtype BAM SortedByCoordinate --outFileNamePrefix "$ID"_

# 2. SAM to sorted BAM
# -F 260 filters out unmapped and secondary alignments
samtools view -@ 30 -Sb -F 260 "$ID"_Aligned.sortedByCoord.out.bam > "$ID"_Aligned.sortedByCoord.filtered.bam
samtools index "$ID"_Aligned.sortedByCoord.filtered.bam

featureCounts -T 1 -t exon -g gene_name --fracOverlap 0.25 -a "$GTF" -o "$ID"_ReadCount.featureCounts.gencode.txt "$ID"_Aligned.sortedByCoord.filtered.bam