Representative sequence chromatograms illustrating how mixed STR sequences can be detected by direct sequencing. (A) Partial sequence chromatograms of MG309-STRs in specimen no. 57 which contained a mixture of 12 and 11 repeats. (B) Partial sequence chromatograms of MG309-STRs in specimen no. 198.2 which contained a mixture of 15, 16 and 17 repeats. The PCR product from each specimen was directly sequenced in two separate sequencing reactions from forward and reverse directions, respectively. The AGT/AAT repeat units are underlined. In direct sequencing of the PCR products, the minority population (11 repeats in panel A and 16 and 17 repeats in panel B) is three bases out of alignment with the majority population (12 repeats in panel A and 15 repeats in panel B), resulting in a mixture of two or three chromatogram peaks as indicated by asterisks. The sequences flanking the repeat region can be determined accurately by sequencing from two directions. The estimated individual sequences shown above the original sequence readouts were confirmed by sequencing of plasmid clones for both specimens (no. 57 in additional file 2 and no. 198.2 in additional file 2 and Figure 3B).