Additional files: How are my short read and or fasta files supposed to be formatted or formulated?
Here I will show examples of how to format the additional files, most pertinent for our case, the short-read sequencing files and the fasta-reference.
1. Short-read sequencing:
- Optionally, one can add short-read bulk RNA sequencing data and use it to confirm the existence of the splice site signals.
- If this is desired, then the input file requires preprocessing to: align the raw sequencing reads; remove possible multi-alignment, supplementary reads, and secondary alignment. This preprocessing is similar to how the long-read sequencing input under the Fastq and the SAM subsection.
- Ideally, the input file is a compressed BAM-file (
[Short-read-Input.bam]) formated as following:
2. Fasta reference file:
- Another additional option is to add the reference genome of your targeted organism for the detection of canonical GU/AG splice site dinucleotides.
- My recommendation is to download the reference file from NCBI and have it in standard formating. Standard formating implies that the headers of the chromosomes/organisms are annotated through the use of the
\>("more than") symbol while the nucleotide sequences themselves are approximately ~108 nucleotides long (105-110 nt):
- If you want to use multichromosomal organism or an amalgamation reference fasta file containing the reference sequence for multiple organisms, then the header needs to be formated as following:
