Attaching Unique Molecular Identifiers (UMI) to RNA molecules in the first step of sequencing library preparation establishes a distinct identity for each input molecule. This makes it possible to eliminate the effects of PCR amplification bias, which is particularly important where many PCR cycles are required, for example, in single cell studies. After PCR, molecules sharing a UMI are assumed to be derived from the same input molecule. In our single cell RNA-Seq studies of Physcomitrella
patens, we discovered that reads sharing a UMI, and therefore presumed to be derived from the same mRNA molecule, frequently map to different, but closely spaced locations. This behaviour occurs in all such libraries that we have produced, and in multiple other UMI-containing RNA-Seq data sets in the public domain. This apparent paradox, that reads of identical origin map to distinct genomic coordinates may be partially explained by PCR stutter, which is often seen in low-entropy templates and those containing simple tandem repeats. In the absence of UMI this artefact is undetectable. We show that the common assumption that sequence reads having different mapping coordinates are derived from different starting molecules does not hold. Unless taken into account, this artefact is likely to result in over-estimation of certain transcript abundances, depending on the counting method employed.
A characteristic feature of a plant immune response is the increase of the cytosolic calcium (Ca2+) concentration following infection, which results in the downstream activation of immune response regulators. The bryophyte Physcomitrella patens has been shown to mount an immune response when exposed to bacteria, fungi, or chitin elicitation, in a manner similar to the one observed in Arabidopsis thaliana. Nevertheless, whether the response of P. patens to microorganism exposure is Ca2+ mediated is currently unknown. Here, we show that P. patens plants treated with chitin oligosaccharides exhibit Ca2+ oscillations, and that a calcium ionophore can stimulate the expression of defense-related genes. Treatment with chitin oligosaccharides also results in an inhibition of growth, which can be explained by the depolymerization of the apical actin cytoskeleton of tip growing cells. These results suggest that chitin-triggered calcium oscillations are conserved and were likely present in the common ancestor of bryophytes and vascular plants.
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