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In our study, the detection of annotation errors, such as incorrect start sites assignment, sequencing errors, and wrongly annotated pseudogenes, would prevent misannotation from being multiplied in future versions of the S. flexneri 2a str.301 genome.
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We handle this uncertainty in our P-site assignment strategy by assigning a fraction of the P-site over a neighborhood of adjacent nucleotides determined by the length and endpoints of each read alignment as detailed in the methods section of our manuscript and as previously performed in Oh et al., 2011.
Random editing site assignment was used to produce coding sequences with randomly assigned editing sites.
Further, all spectra corresponding to phosphopeptides were manually inspected for correct sequence identification validation and site assignment based on the presence of site determining fragment ions.
In the MS/MS spectrum, its phosphorylation site is sandwiched by fragments still bearing the modification as well as the corresponding neutral loss (-98Da) fragments, i.e. y4, y5, y6, y7, y8-98, y8, y9-98, y9, y10, which validates the phosphorylation site assignment.
All the hurdles make accurate O-GlcNAc site assignment a challenging task.
Manual analysis of MS/MS patterns were performed to validate phosphorylation site assignment.
Initial sequence identification and phopsho-site assignment was performed by Sequest (University of Washington).
Oxidation site assignment using our top-down approach was possible for all detected oxidized filgrastim variants.
Figure 2 shows the phospho-site assignment for one of the phosphorylated peptides as an example.
Citrullinated peptides were inspected for validity of site assignment (Additional file 2: Figures S1-S7).
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