(a) D2S441 (b) D19S433 (c) D22S1045 Fusion Fusion Fusion GlobalFiler

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(a) D2S441 (b) D19S433 (c) D22S1045 Fusion Fusion Fusion GlobalFiler Fig. S1 Electropherograms of the silent alleles observed using GlobalFiler® at the D2S441 (a), D19S433 (b), and D22S1045 (c) loci. Each sample was amplified using 0.5 ng of each DNA with PowerPlex® Fusion and GlobalFiler®. The numerals in each box show the allele call, peak height (RFU), and estimated fragment size (bp).

(a) D2S441: allele 12 (U14C→T), forward direction (b) D19S433 Alleles 13, 14.2 (D32G→G, A), reverse direction Y (T, C) D32: A, G +1 -32 Repeat region U14: T -1 -14 (b) D19S433: alleles 13, 14.2 (D32G→G, A), reverse direction Y (T, C) D32: A, G +1 +32 Repeat region (c) D22S1045: allele 12 (U18C→T), forward direction U18: T Repeat region -1 -18 Fig. S2 Electropherograms of the sequencing analysis of silent alleles at the D2S441 (a), D19S433 (b), and D22S1045 (c) loci. The separated allele was analyzed for the D2S441 and D221045 loci (a, c). A total of 30 discordant samples were analyzed for the D19S433 locus without allelic separation, and the sample shown in Fig. S1 is shown as an example (b).

(a) 2800M DNA, with “D2_906_20F” primer (1 pmol) D2S441 locus (a) 2800M DNA, with “D2_906_20F” primer (1 pmol) (b) 2800M DNA, a control without additive primer D2_GF_F & D2_GF_R (NED) D2_906_20F & D2_GF_R (NED) D2_GF_F & D2_GF_R (NED) [ (106.08 – 85.09) + (121.91 – 101.38) ] / 2 = 20.76 ≈ 21 This result indicated that the 5’ end position of GlobalFiler D2S441 forward “D2_GF_F” primer was estimated to be located about 21 nucleotides downstream of that of the “D2_906_20F” primer. (c) -11 -21 -31 -41 U14C→T Repeat Region 5’ end of D2_GF_F D2_906_20F -1 21 nt GRCh38.p2 AAGGGCTACA GGAATCATGA GCCAGGAACT GTGGCTCATC TATGAAAACT TCTATCTATC TATCTATCTA TCTATCTATC TATCTATCTA TCTATCTATA TCATAACACC Allele12(U14C→T) .......... .......... .......... ......T... .......... .......... .......... .......... .......... .......... .......... Fig. S3 Estimation of the 5’ end position of the GlobalFiler® D2S441 forward “D2_GF_F” primer. The “D2_906_20F” primer was added to the reaction mixture of GlobalFiler, and 2800M DNA was amplified (a). As a control, the same DNA was amplified without an additive primer using GlobalFiler (b). The estimated 5’ end position of the GlobalFiler® forward “D2_GF_F” primer and the mutation site “U14C→T” are shown in the aligned sequence of the silent allele toward the NCBI reference sequence (NT_022184.16) contained in the human genome assembly data of GRCh38.p2 (c).

(a) 2800M DNA, with “D22_MiniSTR_F” primer (1 pmol) D22S1045 locus (a) 2800M DNA, with “D22_MiniSTR_F” primer (1 pmol) (b) 2800M DNA, a control without additive primer D22_MiniSTR_F & D22_GF_R (TAZ) D22_GF_F & D22_GF_R (TAZ) D22_GF_F & D22_GF_R (TAZ) 112.54 – 106.78 = 5.76 ≈ 6 This result indicated that the 5’ end position of the GlobalFiler D22S1045 forward “D22_GF_F” primer was estimated to be about 6 nucleotides upstream of that of the “D22_MiniSTR_F” primer. (c) 5’ end of D22_GF_F D22_MiniSTR_F -11 -21 -31 -1 Repeat Region U18C→T 6 nt GRCh38.p2 CTGCTATGGG GGCTAGATTT TCCCCGATGA TAGTAGTCTC ATTATTATTA TTATTATTAT TATTATTATT ATTATTATTA TTACTATTAT TGTTATAAAA Allele12(U18C→T) .......... .......... ..T....... .......... .......... .......... .......--- ---------- --........ .......... Fig. S4 Estimation of the 5’ end position of the GlobalFiler® D22S1045 forward “D22_GF_F” primer. The “D22_MiniSTR_F” primer was added to the reaction mixture of GlobalFiler®, and 2800M DNA was amplified (a). As a control, the same DNA was amplified without an additive primer using GlobalFiler® (b). The estimated 5’ end position of the GlobalFiler® forward “D22_GF_F” primer and the mutation site “U18C→T” are shown in the aligned sequence of the silent allele toward the NCBI reference sequence (NT_011520.13) contained in the human genome assembly data of GRCh38.p2 (c).