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As DNA typing systems have become increasingly sensitive in recent years, probability distribution models for back, forward, double-back, and minus 2-nt stutter ratios have been desired to be considered in DNA evidence interpretation using specific software programs. However, experimental investigations have been insufficient, especially for forward, double-back, and minus 2-nt stutters. In this study, we experimentally reevaluated the probability distribution models for each stutter ratio in the typing systems of GlobalFiler™ PCR Amplification Kit and 3500xL Genetic Analyzer from Thermo Fisher Scientific. In addition, to enhance the reliability of longest uninterrupted stretch (LUS) values and corrected allele numbers used in previously developed models for stutter ratios using sequence information (i.e., LUS model and multi-seq model), we propose the weighted average of LUS values and corrected allele numbers based on the number of observations in sequence-based population data. Back stutter ratios demonstrated a positive correlation with allele numbers (allele model) in eight loci, LUS values (LUS model) in eight loci, and corrected allele numbers (multi-seq model) in five loci. The forward stutter ratios (FSRs) of D22S1045 followed the LUS model. FSRs other than D22S1045 and double-back stutter ratios followed the LUS model by considering multiple loci together. Minus 2-nt stutter ratios observed in SE33 and D1S1656 did not increase with the increase in the allele numbers. The adopted models for each stutter ratio can be implemented in software programs for DNA evidence interpretation and enable a reliable interpretation of crime stain profiles in forensic caseworks.

DYF387S1 is a major Y-chromosome short tandem repeat (Y-STR) used in forensic genetics that is included in the Y-chromosomal haplotype reference database (YHRD, https://yhrd.org) and it is known as a rapidly mutating Y-STR. DYF387S1 is a multi-locus marker and the two paralogs are within a palindromic sequence which is a region prone to structural chromosome mutation. In this study, we investigated DYF387S1 copy number distribution and separately typed the two DYF387S1 paralogs in a Japanese population. We found different DYF387S1 copy numbers among haplogroups indicating that the differences had been caused by haplogroup-specific ancestral Y-chromosomal mutations, such as deletion, duplication and non-allelic gene conversion. In haplogroup C, it is likely that gene conversion between two DYF387S1 paralogs had occurred in the common ancestral Y-chromosome for paragroup C-M130* and duplication of DYF387S1 had occurred in the common ancestral Y-chromosome for haplogroup C-M131. Meanwhile, in haplogroup D, deletion of the upstream DYF387S1 paralog is likely to have occurred in the common ancestral Y-chromosome for paragroup D-M57* and duplication of the remaining DYF387S1 paralog is indicated in the common ancestral Y-chromosome for haplogroup D-M125. In haplogroup O, structural mutations changing the DYF387S1 copy number had probably not occurred in the common ancestral Y-chromosome. We also suggest that deletion of one DYF387S1 paralog occurred in haplogroup N and that deletion of one DYF387S1 paralog or DYF387S1 gene conversion occurred in haplogroup Q. This is the first study that has separately typed the two DYF387S1 paralogs in a large population dataset. As haplogroups C, D, N, O and Q are also observed in other populations, the ancestral mutation events indicated by this study may have affected DYF387S1 polymorphism in other areas of the world.

Y-chromosomal short tandem repeat (Y-STR) markers have been used for forensic purposes such as kinship analysis of male-linage and detection of a male DNA component in a mixture of male and female DNA. Recently, rapidly mutating Y-STR (RM Y-STR) markers were reported that are expected to help distinguish close male relatives. This study provides data of Y-chromosomal haplotypes for 25 Y-STR markers, including six RM Y-STR markers (DYS576, DYS627, DYS518, DYS570, DYS449 and DYF387S1) typed with the Yfiler™ Plus kit in 1299 males of the Japanese population. Discrimination capacity increased from 87.2% for 16 Y-STR markers with the Yfiler™ kit to 99.6% for 25 Y-STR markers with the Yfiler™ Plus kit. We characterized sequences of observed microvariant alleles of eight Y-STR markers and a low-amplified allele of DYS390 by Sanger sequencing. DYF387S1, a multi-locus Y-STR marker that is located at two positions on the human Y-chromosome, was observed in tri-allelic patterns in 51 of 1299 samples (3.9%) and we found an extremely high frequency of the tri-allelic pattern of DYF387S1 in haplogroup C-M131. We also analyzed Y-STR gene diversity in each haplogroup and its relevance to mutation rates.

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.

Rods and cones are both photoreceptors in the retina, but they are different in many aspects including the light response characteristics and, for example, cell morphology and metabolism. These differences would be caused by differences in proteins expressed in rods and cones. To understand the molecular bases of these differences between rods and cones, one of the ways is to compare proteins expressed in rods and cones, and to find those expressed specifically or dominantly. In the present study, we are interested in proteins in the outer segment (OS), the site responsible for generation of rod- or cone-characteristic light responses and also the site showing different morphology between rods and cones. For this, we established a method to purify the OS and the inner segment (IS) of rods and also of cones from purified carp rods and cones, respectively, using sucrose density gradient. In particular, we were interested in proteins tightly bound to the membranes of cone OS. To identify these proteins, we analyzed proteins in some selected regions of an SDS-gel of washed membranes of the OS and the IS obtained from both rods and cones, with Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using a protein database constructed from carp retina. By comparing the lists of the proteins found in the OS and the IS of both rods and cones, we found some proteins present in cone OS membranes specifically or dominantly, in addition to the proteins already known to be present specifically in cone OS.