ExpressionAnalysisPipeline

Pipeline that receives input of multiple fastq files and returns a tsv file with expression level, Entrez ID, GO term etc for each gene.

Require Tools

• Python3
• Trimmomatic
• HISAT2
• samtools
• stringtie
• mygene(python package)

Pipeline Flow Diagram

Column of Output Data

• RefSeq_ID
• each_FPKM
• each_TPM
• each_Coverage
• Gene_ID
• Chromosome
• Start
• End
• Width
• Strand
• Entrez_Gene_ID
• Description
• Cellular_Component_Accession
• Cellular_Component_Name
• Molecular_Function_Accession
• Molecular_Function_Name
• Biological_Process_Accession
• Biological_Process_Name

Output Example

Input Files

Annotation Files

• You have to specify either Fasta File or build completed HISAT2 index files.
• You must enter the gtf / gff file.

Trimmomatic Adapter Path.

• You must set conf.yml in the same directory and describe the paths of Trimmomatic path and adapter path in it.

TRIMMOMATIC_PATH : /usr/local/bioinfo_tools/Trimmomatic-0.36/trimmomatic-0.36.jar
TRIM_ADAPTER_SE : /usr/local/bioinfo_tools/Trimmomatic-0.36/adapters/single_end.fa
TRIM_ADAPTER_PE : /usr/local/bioinfo_tools/Trimmomatic-0.36/adapters/paired_end.fa

Usage

From Fastq

Differences Between Each Script

• expression_analysis.py
  • Process all isoforms.
• expression_analysis_longest.py
  • Process the longest isoform.

Fastq Files

• Multiple single-end · paired-end files can be received at once.
• Avaliable extensions are .fq , .fastq , .fq.gz .
• In the case of paired-end, connect two file paths with a comma.

Help Message

$ python3 expression_analysis.py --help
usage: expression_analysis [-h] [-a FASTA | -i INDEX] -g GTF [-o OUTPUT]
                           [-p CPU]
                           fastq [fastq ...]

Expression analysis pipeline created by suecharo.

positional arguments:
  fastq       Input all fastq file.(.fastq or .fa or .fa.gz) (single-
              end=SRR000001.fq, paired-end=SRR000002_1.fq,SRR000002_2.fq)

optional arguments:
  -h, --help  show this help message and exit
  -a FASTA    Input fasta file of .fa or .fasta.
  -i INDEX    Input HISAT2 index path.(e.g. ./hisat2-index/hg19)
  -g GTF      Input gtf file of hg19 or mm9.
  -o OUTPUT   Enter output dir.(default=./output)
  -p CPU      Input cpu num.(default=4)

Execution Command Example

$ python3 expression_analysis.py \
-i /home/suecharo/data/expression_analysis/hisat2_index/hg19 \
-g /home/suecharo/data/expression_analysis/hg19.gtf \
-o /home/suecharo/analysis/expression_analysis/test \
-p 20 \
/home/suecharo/data/expression_analysis/data/SRR951071/SRR951071.fastq \
/home/suecharo/data/expression_analysis/data/SRR951072/SRR951072_1.fastq,/home/suecharo/data/expression_analysis/data/SRR951072/SRR951072_2.fastq

From Bam

Fastq Files

• The directory of the non-sorted bam file is taken as input.
• Perhaps even if it is sorted.

Help Message

$ python3 expression_analysis_bam.py  --help
usage: expression_analysis_bam.py [-h] -g GTF [-o OUTPUT] [-p CPU] BAM_DIR

Expression analysis pipeline created by suecharo.

positional arguments:
  BAM_DIR     Input sorted bam dir.

optional arguments:
  -h, --help  show this help message and exit
  -g GTF      Input gtf file of hg19 or mm9.
  -o OUTPUT   Enter output dir.(default=./output)
  -p CPU      Input cpu num.(default=4)

Execution Command Example

python3 /app/pipeline/ExpressionAnalysisPipeline/expression_analysis_bam.py  \
-g /data/share/reference/ensembl/Mus_musculus.GRCm38.87.v2.gtf \
-p 10 \
-o ./bam_test \
./bam_files