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Combined analysis of bisulfite-converted STR and iSNP targets using MPS and FDSTools. a Workflow of bisulfite sequencing data analysis. Prior to sequencing, the final library file for bisulfite-converted <t>DNA</t> required for FDSTools package was generated based on the pre-library file. Raw bisulfite sequences are processed (quality check, trimming, merging) before FDSTools packages combined with STRNaming algorithm are used for amplicon detection, allele naming and variant calling (including <t>non-methylated</t> DNAm sites and iSNPs). Conversion assurance is conducted prior to evaluation of marker coverage, conversion efficiency, STR/iSNP allele and DNAm. b Output of FDSTools seqconvert tool (1:1 mixture non-methylated and methylated control DNA). For STR targets, CE-based STR alleles, repeat structure based on ISFG recommendations (red) and flanking variants (blue), including non-methylated DNAm sites, are listed. TDMP-iSNP targets are treated as microhaplotypes and the output contains lined up bases at each CpG within amplicon (blue), location and transition of flanking variants, including alternative alleles at iSNP sites (orange). Column “total” provides the number of reads for each unique sequence and read counts in forward or reverse columns report the amplified bisulfite converted DNA strand. c Based on FDSTools output, read counts of STR (red) and iSNP (orange) genotypes, methylated and non-methylated reads at DNAm sites (blue) can be extracted. R: Guanine or adenine
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Combined analysis of bisulfite-converted STR and iSNP targets using MPS and FDSTools. a Workflow of bisulfite sequencing data analysis. Prior to sequencing, the final library file for bisulfite-converted DNA required for FDSTools package was generated based on the pre-library file. Raw bisulfite sequences are processed (quality check, trimming, merging) before FDSTools packages combined with STRNaming algorithm are used for amplicon detection, allele naming and variant calling (including non-methylated DNAm sites and iSNPs). Conversion assurance is conducted prior to evaluation of marker coverage, conversion efficiency, STR/iSNP allele and DNAm. b Output of FDSTools seqconvert tool (1:1 mixture non-methylated and methylated control DNA). For STR targets, CE-based STR alleles, repeat structure based on ISFG recommendations (red) and flanking variants (blue), including non-methylated DNAm sites, are listed. TDMP-iSNP targets are treated as microhaplotypes and the output contains lined up bases at each CpG within amplicon (blue), location and transition of flanking variants, including alternative alleles at iSNP sites (orange). Column “total” provides the number of reads for each unique sequence and read counts in forward or reverse columns report the amplified bisulfite converted DNA strand. c Based on FDSTools output, read counts of STR (red) and iSNP (orange) genotypes, methylated and non-methylated reads at DNAm sites (blue) can be extracted. R: Guanine or adenine

Journal: International Journal of Legal Medicine

Article Title: Linking STRs/SNPs and DNA methylation using massively parallel sequencing for potential forensic applications

doi: 10.1007/s00414-025-03602-2

Figure Lengend Snippet: Combined analysis of bisulfite-converted STR and iSNP targets using MPS and FDSTools. a Workflow of bisulfite sequencing data analysis. Prior to sequencing, the final library file for bisulfite-converted DNA required for FDSTools package was generated based on the pre-library file. Raw bisulfite sequences are processed (quality check, trimming, merging) before FDSTools packages combined with STRNaming algorithm are used for amplicon detection, allele naming and variant calling (including non-methylated DNAm sites and iSNPs). Conversion assurance is conducted prior to evaluation of marker coverage, conversion efficiency, STR/iSNP allele and DNAm. b Output of FDSTools seqconvert tool (1:1 mixture non-methylated and methylated control DNA). For STR targets, CE-based STR alleles, repeat structure based on ISFG recommendations (red) and flanking variants (blue), including non-methylated DNAm sites, are listed. TDMP-iSNP targets are treated as microhaplotypes and the output contains lined up bases at each CpG within amplicon (blue), location and transition of flanking variants, including alternative alleles at iSNP sites (orange). Column “total” provides the number of reads for each unique sequence and read counts in forward or reverse columns report the amplified bisulfite converted DNA strand. c Based on FDSTools output, read counts of STR (red) and iSNP (orange) genotypes, methylated and non-methylated reads at DNAm sites (blue) can be extracted. R: Guanine or adenine

Article Snippet: We performed bisulfite sequencing of a 1:1 mixture of non-methylated and methylated control DNA (Human Methylated and Non-methylated DNA Set, Zymo Research) and display FDSTools output of exemplary targets in Fig. b. D16S539 consists of alleles 11 and 14 with the repeat pattern GATA[11] and GATA[14].

Techniques: Methylation Sequencing, Sequencing, Generated, Amplification, Variant Assay, Methylation, Marker, Control