7 Quick Example

Intoduction

• Experiment: E-GEOD-48829

• Species: Escherichia coli

• Assumptions: iRAP was installed and configured.

• All analysis should take less than two hours to run.

Setup the data

• Create the directories to hold the data in the data sub-folders

  mkdir -p $IRAP_DIR/data/reference/ecoli_k12
  mkdir -p $IRAP_DIR/data/raw_data/ecoli_k12

• Put the genome and annotation files in the respective species folder

  cd $IRAP_DIR/data/reference/ecoli_k12
  wget -c ftp://ftp.ensemblgenomes.org/pub/release-37/bacteria/fasta/bacteria_122_collection/escherichia_coli_k_12_gca_000981485/dna/Escherichia_coli_k_12_gca_000981485.EcoliK12AG100.dna.chromosome.I.fa.gz
  wget -c ftp://ftp.ensemblgenomes.org/pub/release-37/bacteria/gtf/bacteria_122_collection/escherichia_coli_k_12_gca_000981485/Escherichia_coli_k_12_gca_000981485.EcoliK12AG100.90.gtf.gz
  gunzip -f Escherichia_coli_k_12_gca_000981485.EcoliK12AG100.90.gtf.gz
  # remove the header from the GTF
  cat Escherichia_coli_k_12_gca_000981485.EcoliK12AG100.90.gtf | grep -v "^#" > tmp && mv tmp Escherichia_coli_k_12_gca_000981485.EcoliK12AG100.90.gtf

• Put the FASTQ files in the respective species raw_data folder

  cd $IRAP_DIR/data/raw_data/ecoli_k12
  wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/SRR933983/SRR933983.fastq.gz
  wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/SRR933984/SRR933984.fastq.gz
  wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/SRR933985/SRR933985.fastq.gz
  wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/SRR933989/SRR933989.fastq.gz
  wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/SRR933990/SRR933990.fastq.gz
  wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/SRR933991/SRR933991.fastq.gz
  #  (only some of them to keep the example small)
  #wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/SRR933986/SRR933986.fastq.gz
  #wget ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR933/SRR933988/SRR933988.fastq.gz

Configuration/control file

• Create iRAP’s experiment configuration/control file

  # experiment name
  name=ecoli_ex
  # species
  species=ecoli_k12
  # reference genome
  reference=Escherichia_coli_k_12_gca_000981485.EcoliK12AG100.dna.chromosome.I.fa.gz
  # gtf file
  gtf_file=Escherichia_coli_k_12_gca_000981485.EcoliK12AG100.90.gtf
  #
  user_trans=auto
  # Enable filtering based on quality
  qual_filtering=on
  # Use a contamination data set to filter out reads
  cont_index=no
  # Toplevel directory with the data
  data_dir=$(IRAP_DIR)/data
  mapper=bowtie2
  # some contrasts...
  # GA=Group A
  contrasts=GAvsGB GBvsGA
  GAvsGB=GA GB
  GBvsGA=GB GA
  GA=FA FB FC
  GB=FD FE
  se=FA FB FC FD FE
  FA=SRR933983.fastq.gz
  FA_rs=50
  FA_qual=33
  FB=SRR933984.fastq.gz
  FB_rs=50
  FB_qual=33
  FC=SRR933985.fastq.gz
  FC_rs=50
  FC_qual=33
  FD=SRR933989.fastq.gz
  FD_rs=50
  FD_qual=33
  FE=SRR933990.fastq.gz
  FE_rs=50
  FE_qual=33
  FF=SRR933990.fastq.gz
  FF_rs=50
  FF_qual=33

Running iRAP

• Its assumed that the configuration file was named ecoli_example.conf

• Dryrun to validate the configuration file and see all commands that will be executed (-n option)

irap conf=ecoli_example.conf mapper=hisat2 de_method=deseq max_threads=8 -n

• Run iRAP to process the experiment

irap conf=ecoli_example.conf mapper=hisat2 de_method=deseq max_threads=8

• Output files

  • Filtered FASTQ files

ls ecoli_ex/irap_qc/*.f.fastq.gz

• Bam files

ls ecoli_ex/irap_qc/hisat2/*.bam

• Gene level quantification

ls ecoli_ex/irap_qc/hisat2/htseq2/genes.raw.htseq2.tsv

• Transcript/isoform level quantification

ls ecoli_ex/irap_qc/hisat2/htseq2/transcripts.raw.htseq2.tsv

• Exon level quantification

ls ecoli_ex/irap_qc/hisat2/htseq2/exons.raw.htseq2.tsv

• Differential expression

ls ecoli_ex/irap_qc/hisat2/htseq2/deseq/*.genes_de.tsv

• Run iRAP to process the experiment with a different set of methods

irap conf=ecoli_example.conf mapper=hisat2 quant_method=htseq2 de_method=deseq2 max_threads=8

irap conf=ecoli_example.conf mapper=none quant_method=kallisto de_method=deseq2 max_threads=8