In eukaryotic non-reference transcriptome sequencing, can the sequence files predicted by ORF be used for primer design without a reference?
In eukaryotic de novo transcriptome sequencing, ORF predicted sequences can provide preliminary guidance for primer design, but further validation is often required. It is recommended to combine transcriptome assembly tools (such as Trinity, SPAdes) to enhance transcript background information and verify the accuracy of ORFs through database alignment (such as NR, Swiss-Prot) to ensure primer specificity and amplification efficiency. Additionally, if there are functional or conserved regions of specific target genes, it is best to optimize primer design based on this to improve primer stability and amplification specificity.
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