Systematic Evaluation of a Novel 6-dye Direct and Multiplex PCR-CE-Based InDel Typing System for Forensic Purposes

Insertion/deletion (InDel) polymorphisms, combined desirable characteristics of both short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), are considerable potential in the fields of forensic practices and population genetics. However, most commercial InDel kits designed based on non-Asians limited extensive forensic applications in East Asian (EAS) populations. Recently, a novel 6-dye direct and multiplex PCR-CE-based typing system was designed on the basis of genome-wide EAS population data, which could amplify 60 molecular genetic markers, consisting of 57 autosomal InDels (A-InDels), 2 Y-chromosomal InDels (Y-InDels), and Amelogenin in a single PCR reaction and detect by capillary electrophoresis, simultaneously. In the present study, the DNA profiles of 279 unrelated individuals from the Hainan Li group were generated by the novel typing system. In addition, we collected two A-InDel sets to evaluate the forensic performances of the novel system in the 1,000 Genomes Project (1KG) populations and Hainan Li group. For the Universal A-InDel set (UAIS, containing 44 A-InDels) the cumulative power of discrimination (CPD) ranged from 1–1.03 × 10–14 to 1–1.27 × 10–18, and the cumulative power of exclusion (CPE) varied from 0.993634 to 0.999908 in the 1KG populations. For the East Asia-based A-InDel set (EAIS, containing 57 A-InDels) the CPD spanned from 1–1.32 × 10–23 to 1–9.42 × 10–24, and the CPE ranged from 0.999965 to 0.999997. In the Hainan Li group, the average heterozygote (He) was 0.4666 (0.2366–0.5448), and the polymorphism information content (PIC) spanned from 0.2116 to 0.3750 (mean PIC: 0.3563 ± 0.0291). In total, the CPD and CPE of 57 A-InDels were 1–1.32 × 10–23 and 0.999965, respectively. Consequently, the novel 6-dye direct and multiplex PCR-CE-based typing system could be considered as the reliable and robust tool for human identification and intercontinental population differentiation, and supplied additional information for kinship analysis in the 1KG populations and Hainan Li group.

Spread of clonal genovar E Chlamydia trachomatis among men who have sex with men

In a previous study, we developed a Multi-Locus VNTRs Analysis (MLVA) typing system, called MLVA-5, for the discrimination of Chlamydia trachomatis genovar E strain. The results suggested the clonal spread of a MLVA-5 type 21 strain among men who have sex with men (MSM). We applied the MLVA-5 typing method on 157 French anorectal genovar E specimens and 19 Swedish specimens collected between 2010 and 2015. A total of 29 MLVA-5 types was obtained, with three predominant types among French samples: 78 specimens belonged to MLVA-5 type 21, two other types, 11 and 13, included 9 and 14 specimens, respectively. In 15 cases, one unique MLVA-5 type was observed for a single patient, 7 of which were new types not previously described. The distribution of MLVA-5 types according to sexual orientation showed that the 7 anorectal specimens from heterosexual patients belonged to 6 genotypes, and the 12 anorectal specimens from bisexual patients comprised eight types. The 95 anorectal specimens from MSM were distributed into 22 types, but 55 (57.9%) of them belonged to MLVA-5 type 21. Among the Swedish specimens from MSM, eight were from MLVA-type 21 (4 urines and 4 anorectal specimens). The results support the hypothesis of the spread of clonal genovar E strain among MSM.

Multi-Locus Sequence Typing of Ehrlichia chaffeensis Reveals Extensive Genotypic Variation across the United States

ABSTRACTEhrlichia chaffeensis causes human monocytic ehrlichiosis, and its principal vector is the Amblyomma americanum tick. The most frequently identified cases of ehrlichiosis come from the southeastern and south central states of the United States. In this study, a molecular typing system was developed that allows for the genetic differentiation of E. chaffeensis isolates. This multi-locus typing system included sequencing and analyzing intergenic regions ECH0033–ECH0035 and ECH0217–ECH0218, plus, variable genes variable length PCR target, 28-kDa, 120-kDa, and hemE. We examined a total of 31 unique isolates from humans and white-tailed deer, and eight DNA samples extracted from infected A. americanum collected from multiple states. This is the largest evaluation of E. chaffeensis isolates and their genotypes. Our findings show that when sequences of all six loci were concatenated and compared, the 39 samples could be separated into 23 genotypes and further grouped into six phylogenetic clades. The data in this study show no clear pattern between the geographic alignment with the genetic differentiation between the strains. As a result, this poses a challenge to understanding the spread of E. chaffeensis in the United States. Interestingly, our findings indicate that multiple strains from distant geographic origins share the same mutations, which suggests that the strains are being moved from one site to another by their hosts or vectors. In addition, we are seeing a northward shift in the lone star tick distribution in the United States. Last, some data also suggest minimal genetic mutations have occurred over time among strains that are within geographical proximity.

Development and validation of a multiplex 19 X-chromosomal short tandem repeats typing system for forensic purposes

X-chromosome short tandem repeat (X-STR) markers are a powerful complementary system used for paternity and forensic casework. This study presents the development and validation of a new highly efficient multiplex-fluorescent-labeled 19 X-STR typing system, including DXS10079, DXS101, DXS10135, DXS10162, DXS6795, DXS6800, DXS6803, DXS6807, DXS6809, DXS6810, DXS7133, DXS7423, DXS981, DXS9902, DXS9907, GATA165B12, GATA172D05, GATA31E08 and HPRTB along with sex-typing locus, amelogenin. The system was validated according to guidelines issued by the Scientific Working Group on DNA Analysis Methods. Allele frequency and forensic parameters were investigated from 1085 (494 males and 591 females) unrelated Beijing Han individuals, the combined power of discrimination by the 19 X-STR loci in females and males, as well as the combined mean exclusion chance in trios and duos, were 0.999999999999999995, 0.99999999995, 0.9999999995, and 0.9999996, respectively. The results demonstrate that this multiplex system is robust and reliable, and considered to be a powerful tool for forensic application.