How to interpret unexpected splicing with STAR?

Overview of Unexpected Splicing in STAR

• Splicing is a crucial RNA processing step that can be studied using RNA-seq data. The STAR (Spliced Transcripts Alignment to a Reference) aligner is a popular choice for detecting splicing events.

 

• Occasionally, unexpected splicing events may be observed. These can include novel splice junctions, aberrant exon skipping, or intron retention.

Initial Setup and Alignment

• Ensure you've installed STAR correctly and have the necessary input files: genome reference, annotation files (GTF format), and RNA-seq reads (FASTQ files).

 

• Use STAR for alignment with parameters set for splicing detection. Typical command: STAR --runThreadN 8 --genomeDir /path/to/genome --readFilesIn /path/to/reads.fastq --outFileNamePrefix /output/path/prefix\_ --outSAMtype BAM SortedByCoordinate

Review STAR Outputs

• Examine the Log.final.out file for alignment statistics, which includes splice junction information. High numbers of uniquely mapped reads relative to multi-mapped reads indicate reliable results.

 

• Check the SJ.out.tab file, which contains splice junctions identified by STAR. This file provides chromosome location, junction start and end, and evidence of annotation support.

Identify Unannotated Junctions

• Compare splice junctions in SJ.out.tab with known annotations using computational tools or custom scripts to distinguish novel junctions from annotated ones.

 

• Focus on junctions with high read support but not present in the reference annotation as potential novel splicing events.

Validate Unexpected Junctions

• Conduct secondary validation with other aligners or alternative splicing prediction tools to confirm novel junction findings.

 

• Cross-reference with experimental data, such as RT-PCR experiments, to provide biological evidence for unexpected splicing events.

Interpret Results in Biological Context

• Integrate splicing event data with gene expression profiles to determine whether novel splicing might impact gene function or regulation.

 

• Consider biological implications, such as altered protein domains or disrupted regulatory elements, which may result from novel splicing patterns.

Report and Document Findings

• Compile a report summarizing novel splice variants, supporting evidence, and potential biological implications. Include figures and tables where appropriate for clarity.

 

• Provide detailed documentation of all parameters and tools used for reproducibility and further analysis by the research community.