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RNAseq variant calling: STAR align promotes mismatched overhanging mappings onto introns instead of calling splicing junctions

Hi, I'm doing variant calling from my RNAseq data on a cell line (Paired End 2X100b, Illumina) I'm following GATK workflows and everything is working almost fine since I can find all the expected mutations already characterized in my cell line. The problem is that I see some additional mutations that are clearly the result of an alignment error (e.g. NM_001364837:exon8:c.727+1G>A, rs781065280) I'm using the latest release of STAR aligner 2.7.6a to perform the alignment with the following options

STAR --runThreadN 8 --genomeDir $GENOME_DIR \
  --readFilesIn ...   --readFilesCommand gunzip -c \
  --outSAMattrRGline ... \
  --outSAMtype BAM SortedByCoordinate \
  --twopassMode Basic \
  --outSAMmapqUnique 60 \
  --outFilterType BySJout --alignIntronMin 20 \
  --alignIntronMax 1000000 --alignMatesGapMax 1000000 \
  --alignSJoverhangMin 8 --alignSJDBoverhangMin 3 --scoreGenomicLengthLog2scale 0 \
  --outFileNamePrefix $OUTPUT_DIR/...

The problem resides in STAR mapping some reads with a mismatching overhang of 4 bp at beginning of the intron instead of picking a perfect match at the beginning of the next exon (see picture attached). enter image description here

As you can see, --alignSJDBoverhangMin is set to 3 and the length of the overhang is 4bp. Also the splice junction should be canonical so a spliced mapping should carry no penalty. Anybody has an explanation for that?

rna-seq star splicing junction
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written by Francesco • Francesco

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