Table 1 Tools, commands, and user-defined parameters in the H³NGST pipeline

Prefetch

prefetch SRR_ID

Default

Fasterq-dump

fasterq-dump –split-files -O $FASTQ_DIR $SRA_FILE

Default

1st FastQC

fastqc -o $QC_DIR $FASTQ_DIR/${SRA_FILE}.fastq

Default

Trimmomatic

trimmomatic [SE/PE] -phred33 ... ILLUMINACLIP:adapters.fa:2:30:10 SLIDINGWINDOW:4:10 MINLEN:20

Default

2nd FastQC

fastqc -o $QC_DIR TRIMMED.fastq

Default

Bwa mem

bwa mem $REFERENCE_GENOME TRIMMED.fastq [or paired files] > aligned.sam

Reference genome

(user-defined)

Samtools

samtools view -Sb aligned.sam | samtools sort -o aligned_sorted.bam | samtools index aligned_sorted.bam

Default

Bedtools

bedtools bamtobed -i aligned_sorted.bam > aligned.bed

Default

Deeptools

bamCoverage -b aligned_sorted.bam -o output.bw –extendReads 200 –binSize 5 –normalizeUsing None

Default

MakeTagDirectory

makeTagDirectory $TAG_DIR aligned.bed -format bed

Default

FindPeaks

findPeaks $TAG_DIR -style STYLE -o auto -fdr FDR [-i CONTROL_TAG_DIR]

STYLE, FDR threshold

(user-defined)

FindMotifsGenome.pl

findMotifsGenome.pl peaks.txt GENOME motif_dir -size 200 -len 8,10,12

Reference genome

(user-defined)

AnnotatePeaks.pl

annotatePeaks.pl peaks.txt GENOME > annotated.txt

Reference genome, Promoter region (user-defined)