An ambiguous sequence-based allele of SE33

The upcoming next-generation sequencing (NGS) technology has revolutionized forensic DNA analysis. However, the good advantages of this technology came with the challenge of interpreting NGS data. As the NGS produced more complex data than the conventional fragment analysis, a new locus designation and allele nomenclature have been established [] to support the interpretation of NGS data for the community and to be backward compatible with the original capillary electrophoresis. These interpretation guidelines and STR sequence structure catalogs worked efficiently with all the STR markers. However, some markers had reported the discrepancy between NGS and capillary electrophoresis (CE) that stemmed from sequence variation [].

SE33 was the most polymorphic short tandem repeat (STR) marker. For NGS technology, this marker was coamplified with Forenseq™ Signature Prep Kit (Verogen) but was not analyzed by Universal Analysis Software (Verogen) []. The flanking region sequence was essentially included with reported sequences since there were variations in these regions that potentially led to discordance results with CE or length-based data []. In 2018, Borsuk and colleagues [] established a motif ID for the common motif pattern of SE33 in a large group of populations. The common motif pattern included 15 bp of 5′ flanking region, repeat region and 24 bp of 3′ flanking region that covered all observed variants. The larger allele displayed a more complex microvariant allele (x.2) with the interruption of TT or CT in the repeat region.

In this study, we demonstrated an ambiguous sequence-based allele of SE33. An allele number of 16.3 was reported in one sample by STRait Razor 3.0 []. However, the repeat region sequence cannot be designated as allele 16.3. Therefore, we investigated further and compared this ambiguous sequence with other previous studies.

SE33 locus showed an allele number of 16.3 showed the highest read count (2070 reads) and allele 27.2 was the second high read count (1174 reads). This heterozygous genotype (16.3, 27.2) was confirmed by capillary electrophoresis. The sequence allele 16.3 was presented using the STRait Razor 3.0 with 106 bp sequence string of 5′- CT CTTT CTTT CTTT C [CTTT]3 C [CTTT]14 __CTTT CTTT CTTT CT CTTT CTTT - 3′ (Bold letter indicated repeat region and __ was a two bp deletion). According to Fig. 1, the repeat motif of [CTTT]3 and [CTTT]14 was interrupted with C and the missing of CT at the beginning of 3′ flanking region was found. This allele number of 16.3 was demonstrated in the STRseq catalog [] (accession no. MH232723.1) with a 106 bp sequence string identical to our finding. According to the supplement materials of [], the same allele was identified as B2 and the allele designation for this motif ID was N + 2.3 when N was 14 (Fig. 1). However, when considering only the sequence of repeat region, [CTTT]3 C [CTTT]14 appeared to be 17.1 rather than 16.3 (Fig. 1). The motif ID system from [] was versatile for SE33 allele designation, especially from NGS data, and provided backward compatibility to the capillary electrophoresis data. SE33 was marked as German core loci [8] and had been expected to be widely used outside of Europe shortly []. B2 was found at 0.24% in global population []. The finding in this study illustrated an ambiguous sequence-based allele from SE33 that could cause discordance between NGS and capillary electrophoresis. The length-based allele and sequence-based allele were potentially gone in different directions. Furthermore, as the complexity of SE33 allele designation was far more than other markers, the interpretation of NGS data should not rely on repeat region alone, and the motif ID system must be studied prior to allele calling.

SE33 was a coamplified STR marker found in Forenseq™ Signature Prep Kit (Verogen) and can be analyzed by several available software that made SE33 more easily studied by NGS technology. However, some cautions need to be considered. For example, the interpretation should not rely on repeat region alone, and the motif ID system needs to be considered prior to allele calling.

This study was supported by the Forensic Genetics Research Unit, Ratchadapiseksompotch Fund, Department of Forensic Medicine, Faculty of Medicine, Chulalongkorn University and keep, Bangkok, Thailand.

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