How to prepare annotations

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

General usage

If you are not interested in periodicity estimation, you only need reference annotations (GTF), the ribosomal Bowtie 2 index, and the STAR index. Protocol-specific or general adapter sequences to be removed are also required for Flexbar.

To prepare the Bowtie2 index

bowtie2-build-s [options] ribosomal_fasta ribosomal_index

where ribosomal_fasta and ribosomal_index must match the values from the configuration file.

To prepare the STAR index

STAR --runMode genomeGenerate [options] --genomeDir star_index --genomeFastaFiles fasta

where fasta and star_index must match the values from the configuration file. See How to add a config file.

For periodicity estimation (default), a BED12+ file with annotated transcripts is also required. You can create this file with

gtf-to-bed12 [options] gtf bed

where gtf is the reference GTF annotation (must match the value from the configuration file), and bed is the BED12+ file to generate. To use this file, it must be placed under the directory specified by genome_base_path and its name must match the pattern <genome_name>.annotated.bed.gz.

Tip

Use Rp-Bp to prepare all annotations and indices at once. Use the same configuration file with the necessary keys to run prepare-rpbp-genome. See How to prepare annotations for more information. This is a must if you want to QC your Ribo-seq data, see Ribo-seq quality control.