Fig. 3: Mobile mRNA identification is not without challenges.

(1) Technological noise can lead to challenges in the assignment of RNA-seq reads. In SNP-based methods of mobile mRNA detection, it is important to be able to differentiate between sequencing-associated errors and genuine SNPs. In the above case, an RNA-seq read with a ‘T’ at the SNP position would be indicative of the read having come from the alternative allele, genotype 2. However, every position has an error rate and the higher the read depth, the more incorrect base calls are to be expected. Base changes could arise for reverse transcriptase or amplification steps, although their error rate is typically orders of magnitude lower than sequencing errors. Conserved regions in gene families can give rise to similar challenges in distinguishing mapping ambiguities from genuine SNPs. Defining an mRNA as being mobile based on thresholds of RNA-seq reads that contain a SNP can result in base-calling errors and mapping ambiguities biasing the interpretation. To reduce the risk of such events occurring, further stringent filters can be applied (for instance, using only SNPs that are bi-allelic²) or applying rigorous statistical comparisons (for instance, estimating the allele calling frequencies and comparing them between homograft and heterograft²¹). (2) Genome complexity and genome quality can lead to mapping challenges. Orthologous sequences (light green) can result in some RNA-seq reads aligning to a different gene and different genotype. Genome assemblies that are not complete (telomere to telomere) from exactly the same genotype as used for grafting can result in potential mismappings. The shaded blue gene in genotype 1 is missing in the reference genome assembly, resulting in RNA-seq reads from this transcript being mapped to genotype 2.