Hi everybody,

after some questions in this forum, I have my STAR parameters ready to map my RNA-Seq data for analysis of alternative splicing and was wondering, if you think this are reasonable STAR settings in order to find alternative exon junctions as reliable and unbiased as possible.

/STAR_folder/STAR --outFilterType BySJout --outFilterMismatchNmax 10 --outFilterMismatchNoverLmax 0.04 --alignEndsType EndToEnd -runThreadN 8 --outSAMtype BAM SortedByCoordinate --alignSJDBoverhangMin 4 --alignIntronMax 300000 --alignSJoverhangMin 8 --alignIntronMin 20 --genomeDir /STAR_index_folder/ --sjdbOverhang 149 --quantMode GeneCounts --sjdbGTFfile /GTF_file_folder/Homo_sapiens.GRCh38.91.gtf --outFileNamePrefix /Output_dir/gn0_1/ --readFilesIn /Input_folder/example.fastq > Epithel_STARaligning.log

The developer of STAR (Alexander Dobin) recommends to do the mapping of a given sample twice, taking the previously found exon junctions as annotated junctions in the second run. So this would be my setting for the first run of mapping.