Fig. 1

ORFik API overview. ORFik is a versatile API for the analysis of translation. It consists of 7 main components with gray arrows representing data flow through the components. Data is first loaded either directly from raw or pre-aligned form or through the Web Data Download API (left blue box). The latter allows for automatic downloading of data which increases replicability by allowing scripts that include the whole process from data retrieval to analysis. If not previously aligned, the Read Aligner API (right blue box) can trim adapters from fastq files, remove contaminants and align the reads, For this, ORFik uses fastp and STAR. Persistent Data (grey box) is then ensured through storage in a ORFik.experiment object which represents an experiment. This contains file paths to NGS data and genome annotation used. From this data can be retrieved to perform Quality Control (left red box). ORFik implements extensive quality control at several processing steps: contamination QC, alignment QC, annotation QC and analysis QC. For analyses, data is typically processed through the TranscriptRanges API (middle red box). This extends GenomicRanges to facilitate work with transcripts, supporting genomically disjoint regions like ORFs spanning several exons (described in Additional file 1: Table S6). At a more high level the Discovery API (right red box) provides tools that take advantage of TranscriptRanges to accomplish common tasks, among others: ORF finding, P-shifting and initiation context analysis over ORFs. Finally, results from the QC and analysis can be displayed through the Visualization API (green box). This supports a range of figures which can be normalized and summarized in a multitude of ways (described in Additional file 1: Table S1)