Ribo-seq Pipeline

Pre-processing and mapping to genome

This pipeline processes fastq files from RiboLACE seq (Immagina Biotech) into BAM files aligned to the human genome.

• Step 1: fastqc (qc of raw fastq files)
• Step 2: cutadapt (read trimming 1)
• Step 3: umi_tools (extract UMIs)
• Step 4: cutadapt (read trimming 2)
• Step 5: bowtie2 (exclusion of rRNA reads)
• Step 6: bowtie2 (exclusion of tRNA reads)
• Step 7: star (alignment to human genome (hg38 by default))
• Step 8: samtools index (required for next step umi_tools dedup)
• Step 9: umi_tools dedup (remove duplicate reads using UMIs)
• Step 10: samtools index (index final bam file)
• Step 11: ribowaltz (creates different qc plots of riboseq data)

Main outputs:
sample.dedup.bam, sample.dedup.bam.bai and ribowaltz pdf files.

Run: ./preprocess.py [-h] [-i] [-j] [-l] [-u] [-r] [-s] [-g] [-a] [-T] sample fastq

positional arguments:
  sample             sample name
  fastq              path to fastq

optional arguments:
  -h, --help         show this help message and exit
  -i, --rrnai    Path to rRNA bowtie2 index prefix (default: refs/rRNA)
  -j, --trnai    Path to tRNA bowtie2 index prefix (default: refs/tRNA)
  -l, --lowlen   lower length filter RiboWaltz (default: 28)
  -u, --uplen    upper length filter RiboWaltz (default: 36)
  -r, --rds      Path to RiboWalzt annotation RDS (default: None)
  -s, --stari    Path to STAR index (default: None)
  -g, --gtf      Path to GTF file (default: None)
  -a, --annotate     optionnal: runs riboWalz create_annotation(gtf) (default:
                     False)
  -T, --threads  Number of threads to use (default: 4)

RibORF

Run: ./riborf.py [-h] [-g] [-p] [-t] [-d] [-T] [-a] sample bam

positional arguments:
  sample                sample name
  bam                   bam output from STAR alignment

optional arguments:
  -h, --help            show this help message and exit
  -g, --genome      Path to genome ref fasta file (default:
                        /ref/GRCh38.primary_assembly.genome.fa)
  -p, --genePred    Path to genePred annotation file (default: /ref/combin
                        ed.gencode.v39.mitranscriptome.unique_id.v2.unannotate
                        d.sorted.genePred)
  -t, --transcriptGenePred 
                        Path to cDNA genePred annotation file (default:
                        /ref/gencode.v39.protein_coding.genePred)
  -d, --readlength  read lengths to consider for readDist step (default:
                        28,29,30,31,32,33,34,35,36)
  -T, --threads     Number of threads to use (default: 30)
  -a, --annotate        optionnal: runs ORF annotate step (default: False)