NSPlex: an efficient method to analyze non-specific peaks amplified using commercial STR kits.

Commercial short tandem repeat (STR) kits exclusively contain human-specific primers; however, various non-human organisms with high homology to the STR kit's primer sequences can cause cross-reactivity. Owing to the proprietary nature of the primers in STR kits, the origins and sequences of most non-specific peaks (NSPs) remain unclear. Such NSPs can complicate data interpretation between the casework and reference samples; thus, we developed "NSPlex", an efficient method to discover the biological origins of NSPs. We used leftover STR kit amplicons after capillary electrophoresis and performed advanced bioinformatics analyses using next-generation sequencing followed by BLAST nucleotide searches. Using our method, we could successfully identify NSP generated from PCR amplicons of a sample mixture of human DNA and DNA extracted from matcha powder (finely ground powder of green tea leaves and previously known as a potential source of NSP). Our results showed our method is efficient for NSP analysis without the need for the primer information as in commercial STR kits.

Y chromosome haplogroup N in a Japanese population is classified into three subclades, and two DYS385 loci, a duplicated Y-STR, are duplicated again in subclade N-M1819.

DYS385 is one of the major Y chromosome short tandem repeats (Y-STRs) in forensic genetics and exists as 2 copies in the human Y chromosome palindrome P4 region. In this study, we found that some samples were estimated to have ≥ 4 copies of DYS385 in Y chromosome haplogroup N in a Japanese population. Y chromosome haplogroup N is distributed widely in eastern/central Asia, Siberia, and eastern/northern Europe, and is also observed in Japan; however, little is known about haplogroup N subclades in the Japanese population. To reveal the link between increased DYS385 copy number and haplogroup N subclades, we sequenced single nucleotide polymorphisms to classify the subclades. As a result, the Japanese Y chromosomes of haplogroup N were classified into three subclades, and an increased DYS385 copy number was specific to subclade N-M1819* (N1b2*). These results are of use in forensic DNA analysis because Y-STR copy number is important for the interpretation of Y-STR typing results of male DNA mixtures and kinship analysis. We also found that DYS458.1 microvariants (DYS458 intermediate alleles with single-nucleotide insertion) were observed only in subclade N-CTS962 (N1b1b∼) samples. Given that previous studies reported that DYS458.1 microvariants are observed in Y chromosomes of haplogroup N in other populations, DYS458.1 might be used to infer haplogroup N subclades without limitation to the Japanese population. The results of this study will be beneficial not only to forensic genetics but also to anthropological studies.

Development and validation of a SYBR green-based mitochondrial DNA quantification method by following the MIQE and other guidelines.

In forensic mitochondrial DNA (mtDNA) analysis, quantitative PCR (qPCR) is usually performed to obtain high-quality sequence data for subsequent Sanger or massively parallel sequencing. Unlike methods for nuclear DNA quantification using qPCR, a calibrator is necessary to obtain mtDNA concentrations (i.e., copies/µL). Herein, we developed and validated a mtDNA quantification method based on a SYBR Green assay by following MIQE [Bustin et al., Clin. Chem. 55 (2009) 611-22] and other guidelines. Primers were designed to amplify nucleotide positions 16,190-16,420 in hypervariable region 1 for qPCR using PowerUp SYBR Green and QuantStudio 5. The optimized conditions were 0.3 µM each primer and an annealing temperature of 60 °C under a 2-step cycling protocol. K562 DNA at 100 pg/µL was converted into a mtDNA concentration of 16,400 copies/µL using linearized plasmid DNA. This mtDNA calibrator was obtained by cloning the synthesized DNA fragments of mtDNA (positions 16,140-16,470) containing a 100-bp inversion. The linear dynamic range of the K562 standard curve was 10,000-0.1 pg/µL (r2 ≥ 0.999). The accuracy was examined using NIST SRM 2372a, and its components A, B, and C were quantified with differences of -29.4%, -35.0%, and -22.0%, respectively, against the mtDNA concentrations calculated from published NIST data. We also examined the specificity of the primers, stability of the reaction mix, precision, tolerance against PCR inhibitors, and cross-reactivity against DNA from various animal taxa. Our newly developed mtDNA quantification method is expected to be useful for forensic mtDNA analysis.

Poly_NumtS_430 or HSA_NumtS_587 observed in massively parallel sequencing of the mitochondrial HV1 and HV2 regions.

An increasing number of studies on massively parallel sequencing of mitochondrial DNA (mtDNA) have been reporting identification of various types of noise or off-target sequences. Herein, we report that an off-target haplotype (sequence length 192 bp) observed in MiSeq data of mtDNA at nucleotide position 16,209-16,400 was likely caused by polymorphic nuclear mitochondrial DNA sequences (NumtS). Buccal DNA samples from Volunteers #001-004 and Control DNA 007 were amplified with our multiplex system of the B (15,998-16,172), C (16,209-16,400), and E (30-289) regions using 2000 copies of mtDNA. A sample index was added using a Nextera XT index kit, and MiSeq Reagent Nano Kit v2 was used in 2 × 251 cycles on a MiSeq FGx. FASTQ files were analyzed by CLC Genomics Workbench 21.0.3. The extracted SAM files were analyzed using our original Excel macro to sum up the read counts as the phased variant calls for each region. An off-target haplotype differing at 19 sites against the revised Cambridge reference sequence was observed in Volunteer #001 (4 in 10 MiSeq data), Volunteer #002 (2 in 9), and Control DNA 007 (6 in 9). In a BLAST search, the sequence of the off-target haplotype matched perfectly to three polymorphic NumtS (Poly_NumtS_430 [KM281528.1], HSA_NumtS_587 [HE613849.1], and nuclear fossil [S80333.1]) and BAC clone of chromosome 11 (AC107937.2). The sequence also matched perfectly to a Filipino mtDNA (KC993973.1) which was inferred as a chimeric sequence of mtDNA and the HSA_NumtS_587. The sequence of the off-target haplotype was not contained in the latest human reference genome sequence (GRCh38.p13). In a phylogenetic tree, the off-target haplotype was genetically distant from modern human mtDNA and not directly connected to them. In conclusion, observed off-target haplotype amplified by our multiplex system was likely caused by Poly_NumtS_430 or HSA_NumtS_587.

Massively parallel sequencing data of 31 autosomal STR loci obtained using the Precision ID GlobalFiler NGS STR Panel v2 for 82 Japanese population samples.

Allele frequencies for 31 autosomal short tandem repeat (STR) loci (CSF1PO, D10S1248, D12ATA63, D12S391, D13S317, D14S1434, D16S539, D18S51, D19S433, D1S1656, D1S1677, D21S11, D22S1045, D2S1338, D2S1776, D2S441, D3S1358, D3S4529, D4S2408, D5S2800, D5S818, D6S1043, D6S474, D7S820, D8S1179, FGA, Penta D, Penta E, TH01, TPOX, and vWA) were obtained using Precision ID GlobalFiler NGS STR Panel v2 from 82 unrelated individuals sampled from the Japanese population. Autosomal STR alleles designated by NGS and conventional capillary electrophoresis were found to be concordant except at D2S441 allele 9.

Frequencies of D19S433 silent alleles in a Japanese population of 1501 individuals and their effect on likelihood ratios calculated in kinship tests.

Although silent alleles in D19S433 typing using the GlobalFiler PCR Amplification Kit have been reported, the exact frequency of the D19S433 silent alleles in population data of 1501 Japanese individuals, which are widely used for the assessment of Japanese STR typing results, is unclear. In this study, we examined the exact D19S433 silent allele frequency in this population data. We newly observed the G32A variant causing silent alleles at D19S433 in five samples. Combining them with data including 30 samples with the variant reported previously, we determined that the total frequency of the silent alleles (i.e. the frequency of the G32A variant) in the 1501 Japanese samples was 0.0117 (35/3002). Using the D19S433 allele frequency data, we evaluated the effect of presence/absence information for the D19S433 silent allele on kinship tests. Likelihood ratios (LRs) were calculated for both simulated parent-child and full sibling cases, revealing that the LR may change by approximately 10-2 to 103 fold when the presence/absence of the D19S433 silent allele is revealed in a kinship test. Therefore, if a sufficiently large or small LR is obtained, there is little need to determine the presence/absence of the D19S433 silent allele in Japanese kinship tests using GlobalFiler. This study will be beneficial for the assessment of Japanese human identification and kinship test results using GlobalFiler.

Development and validation of Kongoh ver. 3.0.1: Open-source software for DNA mixture interpretation in the GlobalFiler system based on a quantitative continuous model.

Probabilistic genotyping software based on continuous models is effective for interpreting DNA profiles derived from DNA mixtures and small DNA samples. In this study, we updated our previously developed Kongoh software (to ver. 3.0.1) to interpret DNA profiles typed using the GlobalFiler™ PCR Amplification Kit. Recently, highly sensitive typing systems such as the GlobalFiler system have facilitated the detection of forward, double-back, and minus 2-nt stutters; therefore, we implemented statistical models for these stutters in Kongoh. In addition, we validated the new version of Kongoh using 2-4-person mixtures and DNA profiles with degradation in the GlobalFiler system. The likelihood ratios (LRs) for true contributors and non-contributors were well separated as the information increased (i.e., larger peak height and fewer contributors), and these LRs tended to neutrality as the information decreased. These trends were observed even in profiles with DNA degradation. The LR values were highly reproducible, and the accuracy of the calculation was also confirmed. Therefore, Kongoh ver. 3.0.1 is useful for interpreting DNA mixtures and degraded DNA samples in the GlobalFiler system.

Evaluation of probability distribution models for stutter ratios in the typing system of GlobalFiler and 3500xL Genetic Analyzer.

As DNA typing systems have become increasingly sensitive in recent years, probability distribution models for back, forward, double-back, and minus 2-nt stutter ratios have been desired to be considered in DNA evidence interpretation using specific software programs. However, experimental investigations have been insufficient, especially for forward, double-back, and minus 2-nt stutters. In this study, we experimentally reevaluated the probability distribution models for each stutter ratio in the typing systems of GlobalFiler™ PCR Amplification Kit and 3500xL Genetic Analyzer from Thermo Fisher Scientific. In addition, to enhance the reliability of longest uninterrupted stretch (LUS) values and corrected allele numbers used in previously developed models for stutter ratios using sequence information (i.e., LUS model and multi-seq model), we propose the weighted average of LUS values and corrected allele numbers based on the number of observations in sequence-based population data. Back stutter ratios demonstrated a positive correlation with allele numbers (allele model) in eight loci, LUS values (LUS model) in eight loci, and corrected allele numbers (multi-seq model) in five loci. The forward stutter ratios (FSRs) of D22S1045 followed the LUS model. FSRs other than D22S1045 and double-back stutter ratios followed the LUS model by considering multiple loci together. Minus 2-nt stutter ratios observed in SE33 and D1S1656 did not increase with the increase in the allele numbers. The adopted models for each stutter ratio can be implemented in software programs for DNA evidence interpretation and enable a reliable interpretation of crime stain profiles in forensic caseworks.

Modeling the minus two base pair stutter ratio of the D1S1656 locus: A sequence-based mixture distribution model.

In this study, we propose a stutter ratio for a minus two base pair stutter (-2bpSR) model of the D1S1656 locus in capillary electrophoresis (CE)-based short tandem repeat (STR) typing. DNA from a total of 108 Japanese individuals was analyzed via massively parallel sequencing to investigate the length of the longest uninterrupted stretch of two base repeat motif (2bpLUS value) within repetitive structures involving the flanking region. Additionally, -2bpSR data was collected using the GlobalFiler Kit on a 3500xL Genetic Analyzer. As a result of sequencing analysis, all alleles were classified into two types by their 2bpLUS values. The -2bpSR differed significantly between the types. Then, we modeled the -2bpSR with a mixture log-normal distribution using the classification of alleles based on the 2bpLUS values. Furthermore, probabilities of the sequence type within each repeat number in the mixture log-normal distribution model were estimated using logistic regression for each of the five major detected populations. This study is expected to enable interpretation of STR typing while considering minus two base pair stutter at the D1S1656 locus.

Differences in DYF387S1 copy number distribution among haplogroups caused by haplogroup-specific ancestral Y-chromosome mutations.

DYF387S1 is a major Y-chromosome short tandem repeat (Y-STR) used in forensic genetics that is included in the Y-chromosomal haplotype reference database (YHRD, https://yhrd.org) and it is known as a rapidly mutating Y-STR. DYF387S1 is a multi-locus marker and the two paralogs are within a palindromic sequence which is a region prone to structural chromosome mutation. In this study, we investigated DYF387S1 copy number distribution and separately typed the two DYF387S1 paralogs in a Japanese population. We found different DYF387S1 copy numbers among haplogroups indicating that the differences had been caused by haplogroup-specific ancestral Y-chromosomal mutations, such as deletion, duplication and non-allelic gene conversion. In haplogroup C, it is likely that gene conversion between two DYF387S1 paralogs had occurred in the common ancestral Y-chromosome for paragroup C-M130* and duplication of DYF387S1 had occurred in the common ancestral Y-chromosome for haplogroup C-M131. Meanwhile, in haplogroup D, deletion of the upstream DYF387S1 paralog is likely to have occurred in the common ancestral Y-chromosome for paragroup D-M57* and duplication of the remaining DYF387S1 paralog is indicated in the common ancestral Y-chromosome for haplogroup D-M125. In haplogroup O, structural mutations changing the DYF387S1 copy number had probably not occurred in the common ancestral Y-chromosome. We also suggest that deletion of one DYF387S1 paralog occurred in haplogroup N and that deletion of one DYF387S1 paralog or DYF387S1 gene conversion occurred in haplogroup Q. This is the first study that has separately typed the two DYF387S1 paralogs in a large population dataset. As haplogroups C, D, N, O and Q are also observed in other populations, the ancestral mutation events indicated by this study may have affected DYF387S1 polymorphism in other areas of the world.

Evaluation of Rapid DNA system for buccal swab and disaster victim identification samples.

An evaluation of a Rapid DNA system was performed using buccal swab samples and mock Disaster Victim Identification (DVI) samples collected postmortem. The allelic ladder success rate was 90% and samples analyzed simultaneously with this allelic ladder were used for further analysis. Sample success rate of the Rapid DNA system for buccal swab samples, and blood and muscle DVI samples were calculated. Success rates of buccal swab samples were 100% and 75% using cassettes preloaded with all reagents suitable for high- and low-DNA content samples, respectively. Success rates of fresh DVI samples were 80% to 100%. Success rates of putrefied DVI samples varied widely between 0% and 20% and 50% to 80% depending on cassette and sample types. Conventional DNA analysis was performed for comparison with the results of the Rapid DNA system. DNA quantity and degradation of human DNA were measured using quantitative polymerase chain reaction. DVI samples that yielded more than 1 ng/µL of DNA when extracted with conventional protocols were suitable for analysis using cassettes for both high- and low-DNA content samples. DVI samples with less than 0.1 ng/µL of DNA were suitable only for analysis using cassettes for low-DNA content samples. All alleles called and exported by the Expert system software implemented in the Rapid DNA system were concordant with allele calls made by conventional capillary electrophoresis DNA analysis.

Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing.

We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209-16,400) and B- (positions 30-284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.

Polymorphisms and microvariant sequences in the Japanese population for 25 Y-STR markers and their relationships to Y-chromosome haplogroups.

Y-chromosomal short tandem repeat (Y-STR) markers have been used for forensic purposes such as kinship analysis of male-linage and detection of a male DNA component in a mixture of male and female DNA. Recently, rapidly mutating Y-STR (RM Y-STR) markers were reported that are expected to help distinguish close male relatives. This study provides data of Y-chromosomal haplotypes for 25 Y-STR markers, including six RM Y-STR markers (DYS576, DYS627, DYS518, DYS570, DYS449 and DYF387S1) typed with the Yfiler™ Plus kit in 1299 males of the Japanese population. Discrimination capacity increased from 87.2% for 16 Y-STR markers with the Yfiler™ kit to 99.6% for 25 Y-STR markers with the Yfiler™ Plus kit. We characterized sequences of observed microvariant alleles of eight Y-STR markers and a low-amplified allele of DYS390 by Sanger sequencing. DYF387S1, a multi-locus Y-STR marker that is located at two positions on the human Y-chromosome, was observed in tri-allelic patterns in 51 of 1299 samples (3.9%) and we found an extremely high frequency of the tri-allelic pattern of DYF387S1 in haplogroup C-M131. We also analyzed Y-STR gene diversity in each haplogroup and its relevance to mutation rates.

Ratios and distances of pull-up peaks observed in GlobalFiler kit data.

Short tandem repeat (STR) analysis is widely used for forensic examinations with a capillary electrophoresis instrument such as the 3500xL Genetic Analyzer. This instrument adapts multi-locus STR kits to examine up to 27 loci using a 6-fluorescent dye system and corrects the spectral overlap between each dye. However, inaccurate spectral correction can cause pull-up peaks. Here, we examined the pull-up peaks observed in GlobalFiler kit data in terms of their peak height ratios and distances from their parent allele peaks when using the 3500xL and the 3130xl Genetic Analyzers. With the 3500xL, 546 pull-up peaks were observed, and their pull-up ratios averaged 1.03 ±â€¯0.32% (range 0.260-2.80%). Of the 546 pull-up peaks, 534 peaks (97.8%) were within ±1 bp from their parent allele peaks. Overall, the pull-up peaks toward adjacent shorter wavelength channels (e.g., from yellow to green) tended to be observed in the left side (shorter bp) of the corresponding parent allele peaks, and the opposite side tendency was observed for those pull-up peaks toward adjacent longer wavelength channels. These tendencies were also observed in the GlobalFiler data generated with the 3130xl and in the data obtained by injecting a J6 matrix standard with LIZ 500 or 600 v2 size standard into the 3500xL and 3130xl. Inspection of raw data revealed that the shift of pull-up peaks from their parent allele peaks was derived from sigmoid, pull-down, or slightly shifted pull-up shapes. Based on the obtained data, we propose a standard for assessment of questionable pull-up peaks.

Non-specific peaks generated by animal DNA during human STR analysis: Peak characteristics and a novel analysis method for mixed human/animal samples.

Forensic human identification (HID) laboratories occasionally encounter non-specific peaks generated by non-human DNA. Casework samples for human short tandem repeat (STR) profiling may be contaminated by animal DNA because of the specific environment or situation from which they were obtained. Validation studies for HID kits have reported that non-specific peaks generated from some animals are observed near the human amelogenin peak. In this study, we first revealed that DNA sequences associated with the non-specific peaks generated from animal DNA differ from one animal family to the other. However, non-specific peaks cannot be analyzed using the remainder of polymerase chain reaction (PCR) products left over from conventional HID kits when human and animal DNA are mixed. To overcome this issue, we have developed a novel analysis method of using non-specific peaks generated from animal DNA in human STR profiling to identify the source of contaminating animal DNA at the family level. The method applied here is termed as blocking PCR, which involves selective animal DNA re-amplification by blocking nontarget human amelogenin DNA amplification using an oligonucleotide probe that specifically binds to human amelogenin using the remaining PCR product from the HID kit. Our data demonstrated that HID and family discrimination among animals that are often encountered in forensic contexts could be performed simultaneously. This study enabled recovery of more information from limited quantities of casework samples contaminated with animal DNA, which would be useful for forensic HID scientists.

Next-generation sequencing analysis of off-ladder alleles due to migration shift caused by sequence variation at D12S391 locus.

In short tandem repeat (STR) analysis, length polymorphisms are detected by capillary electrophoresis (CE). At most STR loci, mobility shift due to sequence variation in the repeat region was thought not to affect the typing results. In our recent population studies of 1501 Japanese individuals, off-ladder calls were observed at the D12S391 locus using PowerPlex Fusion in nine samples for allele 22, one sample for allele 25, and one sample for allele 26. However, these samples were typed as ordinary alleles within the bins using GlobalFiler. In this study, next-generation sequencing analysis using MiSeq was performed for the D12S391 locus from the 11 off-ladder samples and 33 other samples, as well as the allelic ladders of PowerPlex Fusion and GlobalFiler. All off-ladder allele 22 in the nine samples had [AGAT]11[AGAC]11 as a repeat structure, while the corresponding allele was [AGAT]15[AGAC]6[AGAT] for the PowerPlex Fusion ladder, and [AGAT]13[AGAC]9 for the GlobalFiler ladder. Overall, as the number of [AGAT] in the repeat structure decreased at the D12S391 locus, the peak migrated more slowly using PowerPlex Fusion, the reverse strand of which was labeled, and it migrated more rapidly using GlobalFiler, the forward strand of which was labeled. The allelic ladders of both STR kits were reamplified with our small amplicon D12S391 primers and their mobility was also examined. In conclusion, off-ladder observations of allele 22 at the D12S391 locus using PowerPlex Fusion were mainly attributed to a relatively large difference of the repeat structure between its allelic ladder and off-ladder allele 22.