When good transcripts go bad: artifactual RT-PCR 'splicing' and genome analysis

Area of Science:

• Genomics
• Molecular Biology
• Evolutionary Biology

Background:

• Accurate gene and intron prediction is crucial for understanding genome evolution.
• Recent studies on human intron gain have yielded conflicting results, suggesting either no new introns or numerous gains via duplication.
• Repetitive regions pose challenges for identifying genuine intron gains.

Purpose of the Study:

• To investigate the impact of template switching on recent intron gain detection.
• To identify artifactual splicing events caused by reverse transcriptase in cDNA/EST libraries.
• To explain discrepancies in recent intron gain studies in humans.

Main Methods:

• Analysis of transcript sequences from intron-poor organisms.
• Identification of artifactual splicing-like events.
• Comparison of findings with previous genome-wide intron gain studies.

Main Results:

• Large numbers of apparent template switching events were observed in transcript sequences.
• Template switching was particularly prevalent in repetitive regions.
• These artifactual events can mimic genuine intron gains.

Conclusions:

• Template switching is an underappreciated phenomenon that complicates intron gain detection.
• This artifact can lead to erroneous conclusions about gene and genome evolution.
• Further research is needed to differentiate true intron gains from template switching artifacts.