Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylo

An efficient multiplex genotyping approach for detecting the major worldwide human Y-chromosome haplogroups

The Y chromosome is paternally inherited and therefore serves as an evolutionary marker of patrilineal descent. Worldwide DNA variation within the non-recombining portion of the Y chromosome can be represented as a monophyletic phylogenetic tree in which the branches (haplogroups) are defined by at least one SNP. Previous human population genetics research has produced a wealth of knowledge about the worldwide distribution of Y-SNP haplogroups. Here, we apply previous and very recent knowledge on the Y-SNP phylogeny and Y-haplogroup distribution by introducing two multiplex genotyping assays that allow for the hierarchical detection of 28 Y-SNPs defining the major worldwide Y haplogroups. PCR amplicons were kept small to make the method sensitive and thereby applicable to DNA of limited amount and/or quality such as in forensic settings. These Y-SNP assays thus form a valuable tool for researchers in the fields of forensic genetics and genetic anthropology to infer a man's patrilineal bio-geographic ancestry from DNA

Towards simultaneous individual and tissue identification: A proof-of-principle study on parallel sequencing of STRs, amelogenin, and mRNAs with the Ion Torrent PGM

Abstract DNA-based individual identification and RNA-based tissue identification represent two commonly-used tools in forensic investigation, aiming to identify crime scene sample donors and helping to provide links between DNA-identified sample donors and criminal acts. Currently however, both analyses are typically performed separately. In this proof-of-principle study, we developed an approach for the simultaneous analysis of forensic STRs, amelogenin, and forensic mRNAs based on parallel targeted DNA/RNA sequencing using the Ion Torrent Personal Genome Machine® (PGM™) System coupled with the AmpliSeq™ targeted amplification. We demonstrated that 9 autosomal STRs commonly used for individual identification (CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX, and vWA), the AMELX/AMELY system widely applied for sex identification, and 12 mRNA markers previously established for forensic tissue identification (ALAS2 and SPTB for peripheral blood, MMP10 and MMP11 for menstrual blood, HTN3 and STATH for saliva, PRM1 and TGM4 for semen, CYP2B7P1 and MUC4 for vaginal secretion, CCL27 and LCE1C for skin) together with two candidate reference mRNA markers (HPRT1 and SDHA) can all be successfully combined. Unambiguous mRNA-based tissue identification was achieved in all samples from all forensically relevant tissues tested, and STR sequencing analysis of the tissue sample donors was 100% concordant with conventional STR profiling using a commercial kit. Successful STR analysis was obtained from 1 ng of genomic DNA and mRNA analysis from 10 ng total RNA; however, sensitivity limits were not investigated in this proof-of-principle study and are expected to be much lower. Since dried materials with noticeable RNA degradation and small DNA/RNA amplicons with high-coverage sequencing were used, the achieved correct individual and tissue identification demonstrates the suitability of this approach for analyzing degraded materials in future forensic applications. Overall, our study demonstrates the feasibility of simultaneously obtaining multilocus STR, amelogenin, and multilocus mRNA information for combined individual and tissue identification from a small sample of degraded biological material. Moreover, our study marks the first step towards combining many DNA/RNA markers for various forensic purposes to increase the effectiveness of molecular forensic analysis and to allow more forensically relevant information to be obtained from limited forensic material

Identification and characterization of novel rapidly mutating Y-chromosomal short tandem repeat markers

Short tandem repeat polymorphisms on the male‐specific part of the human Y‐chromosome (Y‐STRs) are valuable tools in many areas of human genetics. Although their paternal inheritance and moderate mutation rate (~10−3 mutations per marker per meiosis) allow detecting paternal relationships, they typically fail to separate male relatives. Previously, we identified 13 Y‐STR markers with untypically high mutation rates (>10−2 ), termed rapidly mutating (RM) Y‐STRs, and showed that they improved male relative differentiation over standard Y‐STRs. By applying a newly developed in silico search approach to the Y‐chromosome reference sequence, we identified 27 novel RM Y‐STR candidates. Genotyping them in 1,616 DNA‐confirmed father–son pairs for mutation rate estimation empirically highlighted 12 novel RM Y‐STRs. Their capacity to differentiate males related by 1, 2, and 3 meioses was 27%, 47%, and 61%, respectively, while for all 25 currently known RM Y‐STRs, it was 44%, 69%, and 83%. Of the 647 Y‐STR mutations o

Common DNA variants predict tall stature in Europeans

Genomic prediction of the extreme forms of adult body height or stature is of practical relevance in several areas such as pediatric endocrinology and forensic investigations. Here, we examine 770 extremely tall cases and 9,591 normal height controls in a population-based Dutch European sample to evaluate the capability of known height-associated DNA variants in predicting tall stature. Among the 180 normal height-associated single nucleotide polymorphisms (SNPs) previously reported by the Genetic Investigation of ANthropocentric Traits (GIANT) genome-wide association study on normal stature, in our data 166 (92.2 %) showed directionally consistent effects and 75 (41.7 %) showed nominally significant association with tall stature, indicating that the 180 GIANT SNPs are informative for tall stature in our Dutch sample. A prediction analysis based on the weighted allele sums method demonstrated a substantially improved potential for predicting tall stature (AUC = 0.75; 95 % CI 0.72-0.79) compared to a previous attempt using 54 height-associated SNPs (AUC = 0.65). The achieved accuracy is approaching practical relevance such as in pediatrics and forensics. Furthermore, a reanalysis of all SNPs at the 180 GIANT loci in our data identified novel secondary association signals for extreme tall stature at TGFB2 (P = 1.8 x 10(-13)) and PCSK5 (P = 7.8 x 10(-11)) suggesting the existence of allelic heterogeneity and underlining the importance of fine analysis of already discovered loci. Extrapolating from our results suggests that the genomic prediction of at least the extreme forms of common complex traits in humans including common diseases are likely to be informative if large numbers of trait-associated common DNA variants are available

Toward Male Individualization with Rapidly Mutating Y-Chromosomal Short Tandem Repeats

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysi

Toward Male Individualization with Rapidly Mutating Y-Chromosomal Short Tandem Repeats

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are com- monly used for testing close paternal relationships among individuals and populations, and for male lineage iden- tification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strik- ingly, >99% of the 12,272 unrelated males were com- pletely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836\u20130.9999988). Haplotype sharing between populations was almost ab- sent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in ur- ban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs,29% were differentiated including 27% of the 2,378 father\u2013son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and over- all male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database