Linkage, recombination, and mutation rate analyses of 19 X-chromosomal STR loci in Chinese Southern Han pedigrees.

From Southern Han Chinese samples, we analyzed 19 X-STR markers for linkage, linkage disequilibrium (LD), and mutation rate. The data were collected from two- and three-generation Southern Han Chinese families. These data suggested that both linkage and linkage disequilibrium should be considered while calculating likelihood ratios with X-STR markers in relationship tests. The linkage disequilibrium of these 19 X-STR markers was calculated in our previous research study that was conducted on Southern Han Chinese population. In this study, the recombination fractions between pairs of markers and those obtained from the second-generation Rutgers combined linkage-physical map of the human genome were compared. The observed differences indicated that recombination was not homogeneous along the X chromosome. Therefore, we evaluated the effect on likelihood calculations by referring to haplotype frequencies obtained from allele distributions rather than haplotype counts of Southern Han Chinese population.

Enhanced interrogation of degraded DNA from human skeletal remains: Increased genetic data recovery using the expanded CODIS loci, multiple sex determination markers, and consensus testing.

Skeletal remains are among the most difficult types of samples encountered in forensic DNA casework and historical investigations due to prolonged exposure to environmental insults. DNA extracted from bone often is degraded, in low quantities, and contains co-purified inhibitors from the surrounding soil and/or burial vault material. When sexually dimorphic skeletal elements are not recovered, determining the sex of a decedent can be challenging. With unidentified human skeletal remains, genetic data often are evaluated in concert with anthropological analyses, as well as other types of metadata, to improve confidence in making associations or for positive identifications. This study evaluated a multi-faceted molecular genetic approach to increasing the amount of data that can be recovered from degraded skeletal remains. Results demonstrate that using a newer-generation multiplex (GlobalFiler™) with an expanded set of highly discriminatory DNA markers - combined with co-amplification of three different sex-determining loci, one additional PCR cycle, and testing multiple cuttings from the same bone or multiple regions within a skeleton - can improve reliability and accuracy in skeletal remains identifications by providing data concordance.

Assessment of impact of DNA extraction methods on analysis of human remain samples on massively parallel sequencing success.

Skeletal remains recovered from missing persons' cases are often exposed to harsh environmental conditions resulting in the DNA being damaged, degraded, and/or the samples containing PCR inhibitors. In this study, the efficacy of common extraction methods was evaluated to remove high levels of PCR inhibitors commonly encountered with human remains, and their downstream compatibility with the two leading sequencing chemistries and platforms for human identification purposes. Blood, hair, and bone samples were spiked with high levels of inhibitors commonly identified in each particular substrate in order to test the efficiency of various DNA extraction methods prior to sequencing. Samples were extracted using three commercial extraction kits (DNA IQ™, DNA Investigator, and PrepFiler® BTA), organic (blood and hair only), and two total demineralization protocols (bone only)). Massively parallel sequencing (MPS) was performed using two different systems: Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The overall results showed that all DNA extraction methods were efficient and are fully compatible with both MPS systems. Key performance indicators such as STR and SNP reportable alleles, read depth, and heterozygote balance were comparable for each extraction method. In samples where CE-based STRs yielded partial profiles (bone), MPS-based STRs generated more complete or full profiles. Moreover, MPS panels contain more STR loci than current CE-based STR kits and also include SNPs, which can further increase the power of discrimination obtained from these samples, making MPS a desirable choice for the forensic analysis of such challenging samples.

Increasing the discrimination power of ancestry- and identity-informative SNP loci within the ForenSeq™ DNA Signature Prep Kit.

The use of single nucleotide polymorphisms (SNPs) in forensic genetics has been limited to challenged samples with low template and/or degraded DNA. The recent introduction of massively parallel sequencing (MPS) technologies has expanded the potential applications of these markers and increased the discrimination power of well-established loci by considering variation in the flanking regions of target loci. The ForenSeq Signature Preparation Kit contains 165 SNP amplicons for ancestry- (aiSNPs), identity- (iiSNPs), and phenotype-inference (piSNPs). In this study, 714 individuals from four major populations (African American, AFA; East Asian, ASN; US Caucasian, CAU; and Southwest US Hispanic, HIS) previously reported by Churchill et al. [Forensic Sci Int Genet. 30 (2017) 81-92; DOI: https://doi.org/10.1016/j.fsigen.2017.06.004] were assessed using STRait Razor v2s to determine the level of diversity in the flanking regions of these amplicons. The results show that nearly 70% of loci showed some level of flanking region variation with 22 iiSNPs and 8 aiSNPs categorized as microhaplotypes in this study. The heterozygosities of these microhaplotypes approached, and in one instance surpassed, those of some core STR loci. Also, the impact of the flanking region on other forensic parameters (e.g., power of exclusion and power of discrimination) was examined. Sixteen of the 94 iiSNPs had an effective allele number greater than 2.00 across the four populations. To assess what effect the flanking region information had on the ancestry inference, genotype probabilities and likelihood ratios were determined. Additionally, concordance with the ForenSeq UAS and Nextera Rapid Capture was evaluated, and patterns of heterozygote imbalance were identified. Pairwise comparison of the iiSNP diplotypes determined the probability of detecting a mixture (i.e., observing ≥ 3 haplotypes) using these loci alone was 0.9952. The improvement in random match probabilities for the full regions over the target iiSNPs was found to be significant. When combining the iiSNPs with the autosomal STRs, the combined match probabilities ranged from 6.40 × 10-73 (ASN) to 1.02 × 10-79 (AFA).

Comparative tolerance of two massively parallel sequencing systems to common PCR inhibitors.

Human remains can be severely affected by the environment, and the DNA may be damaged, degraded, and/or inhibited. In this study, a DNA sample (at 1 ng DNA target input in triplicate) was spiked with five concentrations of five inhibitors (humic acid, melanin, hematin, collagen, and calcium) and sequenced with both the HID-Ion AmpliSeq™ Library Kit and ID panel on the Ion PGM™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The objective of this study was to compare the baseline tolerance of the two sequencing chemistries and platforms to common inhibitors encountered in human remains recovered from missing person cases. The two chemistries generally were comparable but not always susceptible to the same inhibitors or at the same capacity. The HID-Ion AmpliSeq™ Library Kit and ID panel and the ForenSeq DNA Signature Prep Kit both were susceptible to humic acid, melanin, and collagen; however, the ForenSeq kit showed greater inhibition to melanin and collagen than the AmpliSeq™ kit. In contrast, the ForenSeq kit was resistant to the effects of hematin and calcium, whereas the AmpliSeq™ kit was highly inhibited by hematin. Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) showed the same trend among inhibitors when using the ForenSeq kit. Generally, locus read depth, heterozygote allele balance, and the numbers of alleles typed were inversely correlated with increasing inhibitor concentration. The larger STR loci were affected more so by the presence of inhibitors compared to smaller STR amplicons and SNP loci. Additionally, it does not appear that sequence noise is affected by the inhibitors. The noise percentage, however, does increase as the inhibitor concentration increases, due to the decrease in locus read depth and not likely because of chemistry effects.

Investigation of the STR loci noise distributions of PowerSeq™ Auto System.

AIM: To characterize the noise and stutter distribution of 23 short tandem repeats (STRs) included in the PowerSeqTM Auto System. METHODS: Raw FASTQ files were analyzed using STRait Razor v2s to display alleles and coverage. The sequence noise was divided into several categories: noise at allele position, noise at -1 repeat position, and artifact. The average relative percentages of locus coverage for each noise, stutter, and allele were calculated from the samples used for this locus noise analysis. RESULTS: Stutter products could be routinely observed at the -2 repeat position, -1 repeat position, and +1 repeat position of alleles. Sequence noise at the allele position ranged from 10.22% to 28.81% of the total locus coverage. At the allele position, individual noise reads were relatively low. CONCLUSION: The data indicate that noise generally will be low. In addition, the PowerSeqTM Auto System could capture nine flanking region single nucleotide polymorphisms (SNPs) that would not be observed by other current kits for massively parallel sequencing (MPS) of STRs.

Massively parallel sequencing of 68 insertion/deletion markers identifies novel microhaplotypes for utility in human identity testing.

Short tandem repeat (STR) loci are the traditional markers used for kinship, missing persons, and direct comparison human identity testing. These markers hold considerable value due to their highly polymorphic nature, amplicon size, and ability to be multiplexed. However, many STRs are still too large for use in analysis of highly degraded DNA. Small bi-allelic polymorphisms, such as insertions/deletions (INDELs), may be better suited for analyzing compromised samples, and their allele size differences are amenable to analysis by capillary electrophoresis. The INDEL marker allelic states range in size from 2 to 6 base pairs, enabling small amplicon size. In addition, heterozygote balance may be increased by minimizing preferential amplification of the smaller allele, as is more common with STR markers. Multiplexing a large number of INDELs allows for generating panels with high discrimination power. The Nextera™ Rapid Capture Custom Enrichment Kit (Illumina, Inc., San Diego, CA) and massively parallel sequencing (MPS) on the Illumina MiSeq were used to sequence 68 well-characterized INDELs in four major US population groups. In addition, the STR Allele Identification Tool: Razor (STRait Razor) was used in a novel way to analyze INDEL sequences and detect adjacent single nucleotide polymorphisms (SNPs) and other polymorphisms. This application enabled the discovery of unique allelic variants, which increased the discrimination power and decreased the single-locus random match probabilities (RMPs) of 22 of these well-characterized INDELs which can be considered as microhaplotypes. These findings suggest that additional microhaplotypes containing human identification (HID) INDELs may exist elsewhere in the genome.

Analysis of Short Tandem Repeat and Single Nucleotide Polymorphism Loci From Single-Source Samples Using a Custom HaloPlex Target Enrichment System Panel.

Short tandem repeats and single nucleotide polymorphisms (SNPs) are used to individualize biological evidence samples. Short tandem repeat alleles are characterized by size separation during capillary electrophoresis (CE). Massively parallel sequencing (MPS) offers an alternative that can overcome limitations of the CE. With MPS, libraries are prepared for each sample, entailing target enrichment and bar coding, purification, and normalization. The HaloPlex Target Enrichment System (Agilent Technologies) uses a capture-based enrichment system with restriction enzyme digestion to generate fragments containing custom-selected markers. It offers another possible workflow for typing reference samples. Its efficacy was assessed using a panel of 275 human identity SNPs, 88 short tandem repeats, and amelogenin. The data analyzed included locus typing success, depth of sequence coverage, heterozygote balance, and concordance. The results indicate that the HaloPlex Target Enrichment System provides genetic data similar to that obtained by conventional polymerase chain reaction-CE methods with the advantage of analyzing substantially more markers in 1 sequencing run. The genetic typing performance of HaloPlex is comparable to other MPS-based sample preparation systems that utilize primer-based target enrichment.

Empirical testing of a 23-AIMs panel of SNPs for ancestry evaluations in four major US populations.

Ancestry informative markers (AIMs) can be used to determine population affiliation of the donors of forensic samples. In order to examine ancestry evaluations of the four major populations in the USA, 23 highly informative AIMs were identified from the International HapMap project. However, the efficacy of these 23 AIMs could not be fully evaluated in silico. In this study, these 23 SNPs were multiplexed to test their actual performance in ancestry evaluations. Genotype data were obtained from 189 individuals collected from four American populations. One SNP (rs12149261) on chromosome 16 was removed from this panel because it was duplicated on chromosome 1. The resultant 22-AIMs panel was able to empirically resolve the four major populations as in the in silico study. Eight individuals were assigned to a different group than indicated on their samples. The assignments of the 22 AIMs for these samples were consistent with AIMs results from the ForenSeq(TM) panel. No departures from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were detected for all 22 SNPs in four US populations (after removing the eight problematic samples). The principal component analysis (PCA) results indicated that 181 individuals from these populations were assigned to the expected groups. These 22 SNPs can contribute to the candidate AIMs pool for potential forensic identification purposes in major US populations.

Selection of highly informative SNP markers for population affiliation of major US populations.

Ancestry informative markers (AIMs) can be used to detect and adjust for population stratification and predict the ancestry of the source of an evidence sample. Autosomal single nucleotide polymorphisms (SNPs) are the best candidates for AIMs. It is essential to identify the most informative AIM SNPs across relevant populations. Several informativeness measures for ancestry estimation have been used for AIMs selection: absolute allele frequency differences (δ), F statistics (F ST), and informativeness for assignment measure (In). However, their efficacy has not been compared objectively, particularly for determining affiliations of major US populations. In this study, these three measures were directly compared for AIMs selection among four major US populations, i.e., African American, Caucasian, East Asian, and Hispanic American. The results showed that the F ST panel performed slightly better for population resolution based on principal component analysis (PCA) clustering than did the δ panel and both performed better than the In panel. Therefore, the 23 AIMs selected by the F ST measure were used to characterize the four major American populations. Genotype data of nine sample populations were used to evaluate the efficiency of the 23-AIMs panel. The results indicated that individuals could be correctly assigned to the major population categories. Our AIMs panel could contribute to the candidate pool of AIMs for potential forensic identification purposes.

An evaluation of the PowerSeq™ Auto System: A multiplex short tandem repeat marker kit compatible with massively parallel sequencing.

Capillary electrophoresis (CE) and multiplex amplification with fluorescent tagging have been routinely used for STR typing in forensic genetics. However, CE-based methods restrict the number of markers that can be multiplexed simultaneously and cannot detect any intra-repeat variations within STRs. Several studies already have indicated that massively parallel sequencing (MPS) may be another potential technology for STR typing. In this study, the prototype PowerSeq(™) Auto System (Promega) containing the 23 STR loci and amelogenin was evaluated using Illumina MiSeq. Results showed that single source complete profiles could be obtained using as little as 62 pg of input DNA. The reproducibility study showed that the profiles generated were consistent among multiple typing experiments for a given individual. The mixture study indicated that partial STR profiles of the minor contributor could be detected up to 19:1 mixture. The mock forensic casework study showed that full or partial profiles could be obtained from different types of single source and mixture samples. These studies indicate that the PowerSeq Auto System and the Illumina MiSeq can generate concordant results with current CE-based methods. In addition, MPS-based systems can facilitate mixture deconvolution with the detection of intra-repeat variations within length-based STR alleles.

Underlying Data for Sequencing the Mitochondrial Genome with the Massively Parallel Sequencing Platform Ion Torrent™ PGM™.

BACKGROUND: Massively parallel sequencing (MPS) technologies have the capacity to sequence targeted regions or whole genomes of multiple nucleic acid samples with high coverage by sequencing millions of DNA fragments simultaneously. Compared with Sanger sequencing, MPS also can reduce labor and cost on a per nucleotide basis and indeed on a per sample basis. In this study, whole genomes of human mitochondria (mtGenome) were sequenced on the Personal Genome Machine (PGMTM) (Life Technologies, San Francisco, CA), the out data were assessed, and the results were compared with data previously generated on the MiSeqTM (Illumina, San Diego, CA). The objectives of this paper were to determine the feasibility, accuracy, and reliability of sequence data obtained from the PGM. RESULTS: 24 samples were multiplexed (in groups of six) and sequenced on the at least 10 megabase throughput 314 chip. The depth of coverage pattern was similar among all 24 samples; however the coverage across the genome varied. For strand bias, the average ratio of coverage between the forward and reverse strands at each nucleotide position indicated that two-thirds of the positions of the genome had ratios that were greater than 0.5. A few sites had more extreme strand bias. Another observation was that 156 positions had a false deletion rate greater than 0.15 in one or more individuals. There were 31-98 (SNP) mtGenome variants observed per sample for the 24 samples analyzed. The total 1237 (SNP) variants were concordant between the results from the PGM and MiSeq. The quality scores for haplogroup assignment for all 24 samples ranged between 88.8%-100%. CONCLUSIONS: In this study, mtDNA sequence data generated from the PGM were analyzed and the output evaluated. Depth of coverage variation and strand bias were identified but generally were infrequent and did not impact reliability of variant calls. Multiplexing of samples was demonstrated which can improve throughput and reduce cost per sample analyzed. Overall, the results of this study, based on orthogonal concordance testing and phylogenetic scrutiny, supported that whole mtGenome sequence data with high accuracy can be obtained using the PGM platform.

High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing.

STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6-200ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250pg of input DNA, and 62pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.

High-quality and high-throughput massively parallel sequencing of the human mitochondrial genome using the Illumina MiSeq.

Mitochondrial DNA typing in forensic genetics has been performed traditionally using Sanger-type sequencing. Consequently sequencing of a relatively-large target such as the mitochondrial genome (mtGenome) is laborious and time consuming. Thus, sequencing typically focuses on the control region due to its high concentration of variation. Massively parallel sequencing (MPS) has become more accessible in recent years allowing for high-throughput processing of large target areas. In this study, Nextera(®) XT DNA Sample Preparation Kit and the Illumina MiSeq™ were utilized to generate quality whole genome mitochondrial haplotypes from 283 individuals in a both cost-effective and rapid manner. Results showed that haplotypes can be generated at a high depth of coverage with limited strand bias. The distribution of variants across the mitochondrial genome was described and demonstrated greater variation within the coding region than the non-coding region. Haplotype and haplogroup diversity were described with respect to whole mtGenome and HVI/HVII. An overall increase in haplotype or genetic diversity and random match probability, as well as better haplogroup assignment demonstrates that MPS of the mtGenome using the Illumina MiSeq system is a viable and reliable methodology.

[Genetic polymorphisms of Investigator Argus X-12 amplification system in Guangdong Han population].

OBJECTIVE: To investigate the genetic polymorphisms of 12 X chromosome short tandem repeat (X-STR) loci of Investigator Argus X-12 amplification kit in Guangdong Han population. METHODS: DNA samples from 200 unrelated individuals (100 males and 100 females) and 103 families (59 father-mother-daughter trios and 44 mother-son duos) were extracted and amplified with fluorescence labeled multiplex PCR system. PCR products were separated and genotyped with capillary array electrophoresis. RESULTS: One hundred and thirty-seven alleles,including 9 off ladder alleles (OL allele) were observed at the 12 X-STR loci in the population. Six mutations were observed in 162 meioses. The combined power of discrimination (DP) was 0.999 999 997 in males and 0.999 999 999 in females, and the combined mean exclusion chance (MEC) was 0.999 999 988 in the trio cases and 0.999 998 013 in the duo cases. CONCLUSION: Investigator-Argus X-12 amplification system is highly polymorphic in Guangdong Han population and it is powerful for personal identification and paternity testing.