How to prepare annotations

Run this once for any given reference genome, as long as the package version and its dependencies remain the same.

General usage

prepare-rpbp-genome [options] config

For all options, consult the API for prepare-rpbp-genome. See also How to add a config file.

Required input

Reference annotations

A reference GTF annotation (with a gtf extension) for your genome, with transcript, exon, and CDS features (start_codon and stop_codon features are not required). The attribute field must include transcript_id and gene_id, and optionally transcript_biotype, gene_name, and gene_biotype. The annotation must match the version of the reference genome sequence.

Caution

The GTF/GFF2 format does not include the stop codon in the terminal exon, i.e. the stop codon is not included in the CDS.

Reference genome sequence

The input FASTA file for your genome including top level sequences, but excluding haplotypes, e.g. the primary assembly file from Ensembl. The identifiers must match those in the GTF annotation file.

Ribosomal sequence

A separate FASTA file with ribosomal DNA (rDNA) sequences which are generally not included in the genome assembly. This is used for in silico rRNA removal. This file can also include other sequences to filter out, .e.g pre-rRNA, tRNA. We typically include the following

  • The large and small ribosomal subunit sequences, e.g. from NCBI.

  • The genomic tRNA sequences e.g. from GtRNAdb.

  • Mt_rRNA, Mt_tRNA and rRNA genes from BioMart. Select options for the “Gene type” filter. For “Attributes”, select “Sequences”, and then specifically “Exon sequences”. Additionally, including the “Gene type” in the header can be helpful for quality control.

Expected output

Output files are written in the BED, FASTA, or TAB-delimited formats.

  • <ribosomal_index>. The Bowtie 2 index.

  • <star_index>. The STAR index.

The base path for the following file is: <genome_base_path>

  • <genome_name>.annotated.bed.gz. Annotated transcripts in BED12+ format.

The base path for the following files is: <genome_base_path>/transcript-index

  • <genome_name>.transcripts.annotated.fa. Annotated transcript sequences in FASTA format.

  • <genome_name>.orfs-genomic[.orf_note].bed.gz. Putative Ribo-seq ORFs in BED12+ format. See More about annotations for details.

  • <genome_name>.orfs-exons[.orf_note].bed.gz. Putative Ribo-seq ORF exons in BED6+ format. The extra columns are exon_index, giving the order of the exon in the transcript, and transcript_start, giving the start position of that index in transcript coordinates.

  • <genome_name>.orfs-labels[.orf_note].tab.gz. Putative Ribo-seq ORF categories and compatible transcripts in TAB-delimited format. See More about annotations for details.

Note

If a de_novo_gtf file is provided, the last three output files are split into annotated and de-novo. The files used by the pipeline, as described above, are the “concatenation” of the respective annotated and de-novo files. In addition, de-novo transcript BED and FASTA files are created. A new GTF file, created by concatenating gtf and de_novo_gtf, is written to genome_base_path. This file may contain repeated features. For mapping with STAR, this is generally not a problem, but it can be for abundance estimation, cf. Ribotools documentation.