title: Next-generation sequencing analysis of off-ladder alleles due to migration shift caused by sequence variation at D12S391 locus.
abstract: In short tandem repeat (STR) analysis, length polymorphisms are detected by capillary electrophoresis (CE). At most STR loci, mobility shift due to sequence variation in the repeat region was thought not to affect the typing results. In our recent population studies of 1501 Japanese individuals, off-ladder calls were observed at the D12S391 locus using PowerPlex Fusion in nine samples for allele 22, one sample for allele 25, and one sample for allele 26. However, these samples were typed as ordinary alleles within the bins using GlobalFiler. In this study, next-generation sequencing analysis using MiSeq was performed for the D12S391 locus from the 11 off-ladder samples and 33 other samples, as well as the allelic ladders of PowerPlex Fusion and GlobalFiler. All off-ladder allele 22 in the nine samples had [AGAT]11[AGAC]11 as a repeat structure, while the corresponding allele was [AGAT]15[AGAC]6[AGAT] for the PowerPlex Fusion ladder, and [AGAT]13[AGAC]9 for the GlobalFiler ladder. Overall, as the number of [AGAT] in the repeat structure decreased at the D12S391 locus, the peak migrated more slowly using PowerPlex Fusion, the reverse strand of which was labeled, and it migrated more rapidly using GlobalFiler, the forward strand of which was labeled. The allelic ladders of both STR kits were reamplified with our small amplicon D12S391 primers and their mobility was also examined. In conclusion, off-ladder observations of allele 22 at the D12S391 locus using PowerPlex Fusion were mainly attributed to a relatively large difference of the repeat structure between its allelic ladder and off-ladder allele 22.
authors: First Department of Forensic Science, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan; Identification Center, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan. Electronic address: fujii@nrips.go.jp.
authors: Koji
authors: K
authors: Fujii
authors: First Department of Forensic Science, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors: Haruhiko
authors: H
authors: Watahiki
authors: First Department of Forensic Science, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors: Yusuke
authors: Y
authors: Mita
authors: Forensic Science Laboratory, Kyoto Prefectural Police Headquarters, 85-3, 85-4 Shimodachiuri-dori, Kamanza-higashiiru, Yabunouchi-cho, Kamigyo-ku, Kyoto 602-8550, Japan.
authors: Yasuki
authors: Y
authors: Iwashima
authors: Identification Center, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors: Hajime
authors: H
authors: Miyaguchi
authors: First Department of Forensic Science, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan; Identification Center, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors: Tetsushi
authors: T
authors: Kitayama
authors: First Department of Forensic Science, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors: Hiroaki
authors: H
authors: Nakahara
authors: First Department of Forensic Science, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors: Natsuko
authors: N
authors: Mizuno
authors: First Department of Forensic Science, National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba 277-0882, Japan.
authors: Kazumasa
authors: K
authors: Sekiguchi
mesh_terms: D000483:Alleles; D014644:Genetic Variation; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
keywords: D12S391; GlobalFiler; Mobility; Next-generation sequencing; PowerPlex Fusion; Short tandem repeat