Abstract
When PCR products are directly sequenced, heterozygous base-calling fluorescence chromatogram data are derived for identifying single nucleotide polymorphisms (SNP), insertion-deletion (Indel), short tandem repeat (STR), and paralogous genes. Indel and STR can be easily detected using the currently available Indelligent or ShiftDetector programs without searching reference sequences. However, the detection of other genomic variants remains a challenge because of the lack of appropriate tools to analyze heterozygous base-calling fluorescence chromatogram data. In this study, we developed the free, web-based “Mixed Sequence Reader (MSR)” that can directly analyze heterozygous base-calling fluorescence chromatogram data in .abi file format with reference sequences. The heterozygous sequences can be identified as two distinct sequences and aligned with reference sequences. Our results showed that MSR may be used for: (i) physically locating Indel and STR sequences by searching the NCBI reference sequences, and determining the copy number of STR, (ii) predicting the combinations of microsatellite pattern using Federal Bureau of Investigation Combined DNA Index System (CODIS), (iii) determining human papilloma virus (HPV) genotypes by searching current viral databases in cases of multiple infections, and (iv) estimating the copy number of paralogous genes, such as β-defensin 4, DEFB4, and its paralog HSPDP3