Applications of massively parallel sequencing in forensic genetics.

Massively parallel sequencing, also referred to as next-generation sequencing, has positively changed DNA analysis, allowing further advances in genetics. Its capability of dealing with low quantity/damaged samples makes it an interesting instrument for forensics. The main advantage of MPS is the possibility of analyzing simultaneously thousands of genetic markers, generating high-resolution data. Its detailed sequence information allowed the discovery of variations in core forensic short tandem repeat loci, as well as the identification of previous unknown polymorphisms. Furthermore, different types of markers can be sequenced in a single run, enabling the emergence of DIP-STRs, SNP-STR haplotypes, and microhaplotypes, which can be very useful in mixture deconvolution cases. In addition, the multiplex analysis of different single nucleotide polymorphisms can provide valuable information about identity, biogeographic ancestry, paternity, or phenotype. DNA methylation patterns, mitochondrial DNA, mRNA, and microRNA profiling can also be analyzed for different purposes, such as age inference, maternal lineage analysis, body-fluid identification, and monozygotic twin discrimination. MPS technology also empowers the study of metagenomics, which analyzes genetic material from a microbial community to obtain information about individual identification, post-mortem interval estimation, geolocation inference, and substrate analysis. This review aims to discuss the main applications of MPS in forensic genetics.

Genetic diversity of the LILRB1 and LILRB2 coding regions in an admixed Brazilian population sample.

Leukocyte immunoglobulin (Ig)-like receptors (LILR) LILRB1 and LILRB2 may play a pivotal role in maintaining self-tolerance and modulating the immune response through interaction with classical and nonclassical HLA molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LILRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next-generation sequencing in a population sample from São Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes, and 41 LILRB2 SNVs arranged into 11 haplotypes. Although we may not exclude as a possible effect of population structure, we found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have revealed that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Overall, our findings demonstrate that LILRB1 and LILRB2 are as polymorphic as HLA class Ib genes and provide strong evidence supporting the directional selection regime hypothesis.

Prediction of eye and hair pigmentation phenotypes using the HIrisPlex system in a Brazilian admixed population sample.

Human pigmentation is a complex trait, probably involving more than 100 genes. Predicting phenotypes using SNPs present in those genes is important for forensic purpose. For this, the HIrisPlex tool was developed for eye and hair color prediction, with both models achieving high accuracy among Europeans. Its evaluation in admixed populations is important, since they present a higher frequency of intermediate phenotypes, and HIrisPlex has demonstrated limitations in such predictions; therefore, the performance of this tool may be impaired in such populations. Here, we evaluate the set of 24 markers from the HIrisPlex system in 328 individuals from Ribeirão Preto (SP) region, predicting eye and hair color and comparing the predictions with their real phenotypes. We used the HaloPlex Target Enrichment System and MiSeq Personal Sequencer platform for massively parallel sequencing. The prediction of eye and hair color was accomplished by the HIrisPlex online tool, using the default prediction settings. Ancestry was estimated using the SNPforID 34-plex to observe if and how an individual's ancestry background would affect predictions in this admixed sample. Our sample presented major European ancestry (70.5%), followed by African (21.1%) and Native American/East Asian (8.4%). HIrisPlex presented an overall sensitivity of 0.691 for hair color prediction, with sensitivities ranging from 0.547 to 0.782. The lowest sensitivity was observed for individuals with black hair, who present a reduced European contribution (48.4%). For eye color prediction, the overall sensitivity was 0.741, with sensitivities higher than 0.85 for blue and brown eyes, although it failed in predicting intermediate eye color. Such struggle in predicting this phenotype category is in accordance with what has been seen in previous studies involving HIrisPlex. Individuals with brown eye color are more admixed, with European ancestry decreasing to 62.6%; notwithstanding that, sensitivity for brown eyes was almost 100%. Overall sensitivity increases to 0.791 when a 0.7 threshold is set, though 12.5% of the individuals become undefined. When combining eye and hair prediction, hit rates between 51.3 and 68.9% were achieved. Despite the difficulties with intermediate phenotypes, we have shown that HIrisPlex results can be very helpful when interpreted with caution.

Applicability of the SNPforID 52-plex panel for human identification and ancestry evaluation in a Brazilian population sample by next-generation sequencing.

SNP analysis is of paramount importance in forensic genetics. The development of new technologies in next-generation sequencing allowed processing a large number of markers in various samples simultaneously. Although SNPs are less informative than STRs, they present lower mutation rates and perform better when using degraded samples. Some SNP systems were developed for forensic usage, such as the SNPforID 52-plex, from the SNPforID Consortium, containing 52 bi-allelic SNPs for human identification. In this paper we evaluated the informativeness of this system in a Brazilian population sample (n = 340). DNA libraries were prepared using a customized HaloPlex Target Enrichment System kit (Agilent Technologies, Inc.) and sequenced in the MiSeq Personal Sequencer platform (Illumina Inc.). The methodology presented here allowed the analysis of 51 out of 52 SNPforID markers. Allele frequencies and forensic parameters were estimated, revealing high informativeness: the combined match probability and power of exclusion were 6.48 × 10-21 and 0.9997, respectively. Population admixture analysis indicates high European contribution (more than 70%) and low Amerindian contribution (less than 10%) in our population, while individual admixture analyses were consistent with the majority of individuals presenting high European contribution. This study demonstrates that the 52-plex kit is suitable for forensic cases in a Brazilian population, presenting results comparable with those obtained using a 16 STR panel.

Extended HLA-G genetic diversity and ancestry composition in a Brazilian admixed population sample: Implications for HLA-G transcriptional control and for case-control association studies.

Human leukocyte antigen-G (HLA-G) is a nonclassical Major Histocompatibility Complex (MHC) molecule with immunomodulatory function and restricted tissue expression. The genetic diversity of HLA-G has been extensively studied in several populations, however, the segment located upstream -1406 has not yet been evaluated. We characterized the nucleotide variation and haplotype structure of an extended distal region (-2635), all exons and the 3'UTR segment of HLA-G by next-generation sequencing (NGS) in a sample of 335 Brazilian individuals. We detected 29 variants at the HLA-G distal promoter region, arranged into 19 haplotypes, among which we identified sites that may influence transcription factor targeting. Although the variation pattern in the distal region resembled the one observed in the conventional promoter segment, molecular signature for balancing selection was observed in the promoter segment from -1406 to -1 (Tajima's D = 2.315, P = 0.017), but not in this distal segment (D = 1.049, P = 0.118). Furthermore, the ancestry composition of this Brazilian population sample was determined by the analysis of SNPforID 34-plex ancestry informative marker (AIM) SNP panel. The distribution of HLA-G haplotypes was ancestry-dependent, corroborating previous findings and emphasizing the importance of considering the ancestry information in association studies.