Ratios and distances of pull-up peaks observed in GlobalFiler kit data

•Ratios and distances of pull-up peaks observed in GlobalFiler data were examined.•Pull-up ratios with the 3500xL averaged 1.03 ± 0.32%, with a range of 0.260–2.80%.•Most of the pull-up peaks were within ±1 bp from their parent allele peaks.•Sigmoid, pull-down, and pull-up shapes were observed in raw data.•We propose a standard for assessment of questionable pull-up peaks. Short tandem repeat (STR) analysis is widely used for forensic examinations with a capillary electrophoresis instrument such as the 3500xL Genetic Analyzer. This instrument adapts multi-locus STR kits to examine up to 27 loci using a 6-fluorescent dye system and corrects the spectral overlap between each dye. However, inaccurate spectral correction can cause pull-up peaks. Here, we examined the pull-up peaks observed in GlobalFiler kit data in terms of their peak height ratios and distances from their parent allele peaks when using the 3500xL and the 3130xl Genetic Analyzers. With the 3500xL, 546 pull-up peaks were observed, and their pull-up ratios averaged 1.03 ± 0.32% (range 0.260–2.80%). Of the 546 pull-up peaks, 534 peaks (97.8%) were within ±1 bp from their parent allele peaks. Overall, the pull-up peaks toward adjacent shorter wavelength channels (e.g., from yellow to green) tended to be observed in the left side (shorter bp) of the corresponding parent allele peaks, and the opposite side tendency was observed for those pull-up peaks toward adjacent longer wavelength channels. These tendencies were also observed in the GlobalFiler data generated with the 3130xl and in the data obtained by injecting a J6 matrix standard with LIZ 500 or 600 v2 size standard into the 3500xL and 3130xl. Inspection of raw data revealed that the shift of pull-up peaks from their parent allele peaks was derived from sigmoid, pull-down, or slightly shifted pull-up shapes. Based on the obtained data, we propose a standard for assessment of questionable pull-up peaks.