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How to interpret unexpected splicing with STAR?

Learn how to analyze unexpected splicing with STAR, including identifying novel junctions, validating findings, and understanding biological implications.

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

 

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