Stutter Peaks Research Articles

Performance comparison of a previously validated microhaplotype panel and a forensic STR panel for DNA mixture analysis

Short Tandem Repeats (STRs) are the most widespread markers in forensic genetics. However, STR stutter peaks can mask alleles from a minor contributor when analysing mixtures, hindering the interpretation of complex profiles. In this study we compared the performance of a previously described panel of microhaplotypes (MHs), an alternative type of forensic marker, against a standard STR kit. The parameters evaluated included: capability of determining the minimum number of contributors in the mixture; percentages of allele drop-outs and drop-ins; retrieval of alleles belonging to the minor contributor, and estimation of likelihood ratio (LR) values. In addition, the capacity of EuroForMix software to estimate each donor’s percentage of contribution was tested, as well as the impact on results when using manually, or automatically prepared libraries. The MH panel showed better performance than STRs for the detection of 2-contributor mixtures, but the lower degree of polymorphism per MH marker hindered the task of deconvolution with multiple contributors. MHs presented higher drop-in rates and lower drop-out rates, a higher capability to recover the minor contributor’s alleles and provided higher LR values than STRs, likely due to the much higher number of loci combined in the panel. Estimations of contributor ratios using EuroForMix showed promising results and marginal differences were found in these values between manually and automatically prepared libraries. Overall, results showed that the mixture detection performance of the MH panel was better or equal to the standard forensic autosomal STR panel, indicating microhaplotypes are informative markers for this purpose.

Validation and Implementation of Long Mononucleotide Repeat Testing for the Detection of Microsatellite Instability: A Single Institution Experience

Introduction: Microsatellites are short, repetitive portions of DNA that are error prone during replication; several mismatch repair proteins recognize and repair these errors. Deficient MMR (dMMR) results in microsatellite instability (MSI), a well-recognized carcinogenic pathway. In Lynch syndrome (LS), germline mutations in MMR genes predispose to colorectal (CRC) and endometrial carcinoma (EC), among others. Immune checkpoint inhibitors (ICI), such as pembrolizumab, are approved to treat dMMR or MSI-high (MSI-H) cancers regardless of tumor type. Therefore, sensitive and specific methods to detect MSI are of critical importance to patient care. Multiple methods are used to detect MSI status, including immunohistochemistry (IHC) for MMR proteins and molecular assays. The Detroit Medical Center utilizes both IHC and PCR-based assays to maximize MSI detection sensitivity. We attempted validation of a new fluorescent PCR-based assay for MSI tumor testing which compares allelic profiles of microsatellite markers from matching normal and tumor samples. The assay utilizes four traditional MSI markers in addition to four long mononucleotide repeat (LMR) markers. LMR markers produce more pronounced fragment length changes, and thus may improve detection of MSI. Methods: Our validation set included 46 carcinoma patient samples with prior PCR-based MSI and MMR IHC testing. These included MSI-high, MSI-low (MSI-L), and microsatellite stable (MSS) cases. DNA was isolated from formalin-fixed, paraffin embedded (FFPE) tissue and amplified. Stutter peak patterns were analyzed, comparing allele sizes and stutter patterns. All cases were reviewed by two directors, with any discrepancies discussed and reviewed to achieve consensus. MSS samples showed identical stutter patterns between tumor and normal pairs. MSI-L samples had a single discrepant marker, whereas MSI-H samples had 2 or more discrepant markers. The results of the LMR assay were compared with the original PCR-based MSI and IHC results to determine test concordance. Results: 42 cases were successfully amplified and included in our results. Fourteen cases were classified as MSI-H by consensus, with a range of 3 to 8 unstable markers out of 8 total markers. IHC results for the MSI-H cases were available in 13/14 cases; thirteen were classified as abnormal. Notably, in one EC MSI-H case, a normal IHC result was reported. Three cases were classified as MSI-L. All were CRC samples, and 2 of 3 were also designated MSI-L using the traditional PCR MSI assay. The remaining MSI-L case was formerly interpreted as MSS. The IHC results for the MSI-L cases were all available and reported as normal. The remaining cases were designated as MSS. IHC results were normal in all but one EC case. Conclusions: Our study validated the LMR MSI Analysis System for detection of MSI; it is now used in clinical practice in our molecular laboratory at DMC, and challenging real-world cases from our assay rollout will be presented graphically. Our validation results demonstrate high concordance (42/42 cases) between the LMR and traditional MSI panels, highlighting the reliability of LMR markers. IHC and PCR results were largely concordant, with occasional discrepancies revealing the importance of a combination approach for MSI detection.

Development and validation of a new multiplex panel using SNaPshot-based DIP-TriSNP markers for forensic DNA mixtures.

Short tandem repeat analysis is challenging when dealing with unbalanced mixtures in forensic cases due to the presence of stutter peaks and large amplicons. In this research, we propose a novel genetic marker called DIP-TriSNP, which combines deletion/insertion polymorphism (DIP) with tri-allelic single nucleotide polymorphism in less than 230bp length of human genome. Based on multiplex PCR and SNaPShot, a panel, including 14 autosomal DIP-TriSNPs and one Y chromosomal DIP-SNP, had been developed and applied to genotyping 102 unrelated Han Chinese individuals in Sichuan of China and simulated a mixture study. The panel sensitivity can reach as low as 0.1ng DNA template, and the minor contributor of DNA can be detected with the highest ratio of 19:1, as indicated by the obtained results. In the Sichuan Han population, the cumulative probability of informative genotypes reached 0.997092, with a combined power of discrimination of 0.999999998801. The panel was estimated to detect more than two alleles in at least one locus in 99.69% of mixtures of the Sichuan Han population. In conclusion, DIP-TriSNPs have shown promising as an innovative DNA marker for identifying the minor contributor in unbalanced DNA mixtures, offering advantages such as short amplifications, increased polymorphism, and heightened sensitivity.

DEPArray™ single-cell technology: A validation study for forensic applications

In forensics investigations, it is common to encounter biological mixtures consisting of homogeneous or heterogeneous components from multiple individuals and with different genetic contributions. One promising mixture deconvolution strategy is the DEPArray™ technology, which enables the separation of cell populations before genetic analysis. While technological advances are fundamental, their reliable validation is crucial for successful implementation and use for casework. Thus, this study aimed to 1) systematically validate the DEPArray™ system concerning specificity, sensitivity, repeatability, and contamination occurrences for blood, epithelial, and sperm cells, and 2) evaluate its potential for single-cell analysis in the field of forensic science. Our findings confirmed the effective identification of different cell types and the correct assignment of successfully genotyped single cells to their respective donor(s). Using the NGM Detect™ Amplification Kit, the average profile completeness for diploid cells was approximately 80%, with ∼ 290 RFUs. In contrast, haploid sperm analysis yielded an average completeness of 51% referring to the haploid reference profile, accompanied by mean peak heights of ∼ 176 RFUs. Although certain alleles of heterozygous loci in diploid cells showed strong imbalances, the overall peak balances yielded acceptable values above ≥ 60% with a mean value of 72% ± 0.21, a median of 77%, but with a maximum imbalance of 9% between heterozygous peaks. Locus dropouts were considered stochastic events, exhibiting variations among donors and cell types, with a notable failure incidence observed for TH01. Within the wet-lab experimentation with >500 single cells for the validation, profiling was performed using the consensus approach, where profiles were selected randomly from all data to better mirror real casework results. Nevertheless, complete profiles could be achieved with as few as three diploid cells, while the average success rate increased to 100% when using profiles of 6–10 cells. For sperms, however, a consensus profile with completeness >90% of the autosomal diploid genotype could be attained using ≥15 cells. In addition, the robustness of the consensus approach was evaluated in the absence of the respective reference profile without severe deterioration. Here, increased stutter peaks (≥ 15%) were found as the main artifact in single-cell profiles, while contamination and drop-ins were ascertained as rare events. Lastly, the technique’s potential and limitations are discussed, and practical guidance is provided, particularly valuable for cold cases, multiple perpetrator rapes, and analyses of homogeneous mixed evidence.

LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instability Detection.

Microsatellites are short tandem repeats of one to six nucleotides that are highly polymorphic and extensively used as genetic markers in numerous biomedical applications, including the detection of microsatellite instability (MSI) in cancer. The standard analytical method for microsatellite analysis relies on PCR amplification followed by capillary electrophoresis or, more recently, next-generation sequencing (NGS). However, their amplification during PCR generates undesirable frameshift products known as stutter peaks caused by polymerase slippage, complicating data analysis and interpretation, while very few alternative methods for microsatellite amplification have been developed to reduce the formation of these artifacts. In this context, the recently developed low-temperature recombinase polymerase amplification (LT-RPA) is an isothermal DNA amplification method at low temperature (32°C) that drastically reduces and sometimes completely abolishes the formation of stutter peaks. LT-RPA greatly simplifies the genotyping of microsatellites and improves the detection of MSI in cancer. In this chapter, we describe in detail all the experimental steps necessary for the development of LT-RPA simplex and multiplex assays for microsatellite genotyping and MSI detection, including the design, optimization, and validation of the assays combined with capillary electrophoresis or NGS.

Genetic research to evaluate plus stutter on D18S51 locus

The PCR amplification of STR loci typically produces a minor byproduct called minus stutter (or plus stutter) that is one repeat unit shorter (or one repeat unit longer) than the original product. These stutters cause some problems that affect the result of STR typing and complicate when comparing results of STR typing. It is necessary to remove the stutter in the forensic STR typing. Minus stutter is filtered out automatically by setting of the analysis software. On the other hand, plus stutter at most loci in STR Kits does not have such setting and it is in particular difficult to distinguish between the original peak and stutter peak.

A survey of the effects of common illicit drugs on forensic DNA analysis

Profiling of DNA associated with illicit drug packages and paraphernalia is a common investigative tool. In addition, research is being conducted regarding the analysis of trace DNA present within illicit drugs and on capsules. The application of trace DNA analysis to illicit drugs has the potential to identify individuals involved in their manufacture and distribution. However, the inhibitory effects of illicit drugs and related compounds on downstream DNA analysis has not yet been investigated. If drug-induced polymerase chain reaction (PCR) inhibition occurs, the quality or informativeness of the resultant DNA profile may be impacted. In this study, the effects of a range of drugs, diluents, adulterants, and synthetic precursors on both quantitative PCR (qPCR) data and short tandem repeat (STR) DNA profiling results were examined. Twenty-two compounds representative of drug compounds and adulterants which may be encountered in drug seizures were spiked with 1 ng/μL and 0.05 ng/μL of control DNA and underwent DNA quantification using Quantifiler™ Trio. A subset of 13 compounds, including the majority that indicated potential inhibition in Quantifiler™ Trio, underwent STR profiling with VeriFiler™ Plus to determine if inhibition also occurred at this stage. The effect of diluting the DNA extract on the extent of inhibition of STR profiling was also investigated. Internal PCR controls within the qPCR were not a reliable indicator of inhibition, although suppression of the short and long autosomal fragments was observed in the presence of many compounds, and four compounds gave inconclusive results. STR internal quality controls indicated inhibition in 5 of the 13 compounds, however, profiles were affected by the presence of 11 of the 13 compounds in various ways such as a decreased average relative fluorescence units (RFU), drop out of certain alleles (some based on allele size range of locus) leading to a decreased likelihood ratio (LR), an increase in the proportion of stutter peaks and the presence of split or shoulder peaks. All profiles improved following a dilution of the compound in the PCR and allowing the generation of LR values in excess of 1 × 1025, indicating inhibition occurred rather than DNA degradation. The data obtained show that removal of some of these compounds is required through an effective DNA extraction process for successful downstream trace DNA profiling. Upon successful PCR, the resultant DNA profiles provide the opportunity for opening new investigative avenues for law enforcement agencies.

Genetic diversity analysis of forty-three insertion/deletion loci for forensic individual identification in Han Chinese from Beijing based on a novel panel.

Due to the virtues of no stutter peaks, low rates of mutation, and short amplicon sizes, insertion/deletion (InDel) polymorphism is an indispensable tool for analyzing degraded DNA samples from crime scenes for human identifications (Wang et al., 2021). Herein, a self-developed panel of 43 InDel loci constructed previously by our group was utilized to evaluate the genetic diversities and explore the genetic background of the Han Chinese from Beijing (HCB) including 301 random healthy individuals. The lengths of amplicons at 43 InDel loci in this panel ranged from 87 to 199 bp, which indicated that the panel could be used as an effective tool to utilize highly degraded DNA samples for human identity testing. The loci in this panel were validated and performed well for forensic degraded DNA samples (Jin et al., 2021). The combined discrimination power (PD) and combined probability of exclusion (PE) values in this panel indicated that the 43 InDel loci could be used as the candidate markers in personal identification and parentage testing of HCB. In addition, population genetic relationships between the HCB and 26 reference populations from five continents based on 19 overlapped InDel loci were displayed by constructing a phylogenetic tree, principal component analysis (PCA), and population genetic structure analysis. The results illustrated that the HCB had closer genetic relationships with the Han populations from Chinese different regions.

A novel set of short microhaplotypes based on non-binary SNPs for forensic challenging samples.

Short tandem repeats (STRs) are the most widely used genetic markers in forensic application, but they are not ideal genetic markers for the analysis of forensic challenging samples such as highly degraded or unbalanced mixed samples because of their relatively large amplicons and stutter peaks. In this study, we developed a set of short microhaplotypes based on non-binary SNPs with molecular extent sizes no longer than 60 bases and genotyped 100 unrelated individuals from northern Han groups. Our results showed this panel has similar discrimination power to STR kits, as the combined random match probability (CMP) reached 1.396 × 10-22 and mean effective number of alleles (Ae) was 3.59. The cumulative probability of exclusion for duos (CPE-duos) was 0.999919 and the cumulative probability of exclusion for trios (CPE-trios) was 0.9999999987, suggesting this panel could be applied for forensic personal identification and parentage testing independently. Population differentiation in 26 populations from the 1000 Genomes Project indicated this panel could distinguish populations from Africa, East Asia, South Asia, America, and Europe. These microhaplotypes based on non-binary SNPs have short amplicons, good discrimination power, no stutter artifacts, and have great potential in detection of highly degraded and unbalanced mixtures for personal identification, paternity testing, and ancestry inference.

High-throughput sequencing-based microsatellite genotyping for polyploids to resolve allele dosage uncertainty and improve analyses of genetic diversity, structure and differentiation: A case study of the hexaploid Camellia oleifera.

Conventional microsatellite (simple sequence repeat, SSR) genotyping methods cannot accurately identify polyploid genotypes leading to allele dosage uncertainty, introducing biases in population genetic analysis. Here, a new SSR genotyping method was developed to directly infer accurate polyploid genotypes. The frequency distribution of SSR sequences was obtained based on deep-coverage high-throughput sequencing data. Corrections were performed accounting for the "stutter peak" and amplification efficiency of SSR sequences. Perl scripts and an online SSR genotyping tool "SSRSeq" were provided to process the sequencing data and output genotypes with corrected allele dosages. Hexaploid Camellia oleifera is the dominant woody oilseed crop in China. Understanding the geographical pattern of genetic variation in wild C. oleifera is essential for the conservation and utilization of genetic resources. Six wild C. oleifera populations were sampled across geographical ranges in subtropical evergreen broadleaf forests of China. Using 35 SSR markers, the high-throughput sequencing-based SSRSeq method was applied to obtain accurate hexaploid genotypes of wild C. oleifera. The results demonstrated that the new method could resolve allele dosage uncertainty and considerably improve genetic diversity, structure and differentiation analyses for polyploids. The genetic variation patterns of wild C. oleifera across geographical ranges agree with the "central-marginal hypothesis", stating that genetic diversity is high in the central population and declines from the central to the peripheral populations, and genetic differentiation increases from the centre to the periphery. This method and findings can facilitate the utilization of wild C. oleifera genetic resources for the breeding of cultivated C. oleifera.

Development and Characterization of Novel Polymorphic Microsatellite Markers for Tapinoma indicum (Hymenoptera: Formicidae).

Tapinoma indicum (Forel) (Hymenoptera: Formicidae) is a nuisance pest in Asia countries. However, studies on T. indicum are limited, especially in the field of molecular biology, to investigate the species characteristic at the molecular level. This paper aims to provide valuable genetic markers as tools with which to study the T. indicum population. In this study, a total of 143,998 microsatellite markers were developed based on the 2.61 × 106 microsatellites isolated from T. indicum genomic DNA sequences. Fifty selected microsatellite markers were amplified with varying numbers of alleles ranging from 0 to 19. Seven out of fifty microsatellite markers were characterized for polymorphism with the Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) analysis. All seven microsatellite markers demonstrated a high polymorphic information content (PIC) value ranging from 0.87 to 0.93, with a mean value of 0.90. There is no evidence of scoring errors caused by stutter peaks, no large allele dropout, and no linkage disequilibrium among the seven loci; although loci Ti-Tr04, Ti-Tr09, Ti-Te04, Ti-Te13, and Ti-Pe5 showed signs of null alleles and deviation from the HWE due to excessive homozygosity. In conclusion, a significant amount of microsatellite markers was developed from the data set of next-generation sequencing, and seven of microsatellite markers were validated as informative genetic markers that can be utilized to study the T. indicum population.

A targeted ancestry informative InDels panel on capillary electrophoresis for ancestry inference in Asian populations.

CE is the primary methodology used in forensic DNA typing. Alleles of commonly used types of genetic markers could be separated and detected via CE based on dye color and migration time. Insertion/deletion (InDel) is an ideal genetic marker for forensic DNA analysis due to their abundance in the human genome, low mutation rate, availability of their allele types via CE, and elimination of stutter peaks. Moreover, InDels could be used as ancestry informative markers since allele frequencies of InDels is different among geographically separated populations. Several ancestry informative insertion/deletion panels have been established based on CE platform to achieve the intercontinental populations distinction. However, improvements to differentiate intracontinental populations is few. In this study, 21 InDels with fixation index (FST ) > 0.15were selected and assembled into one ancestry informative insertion/deletion panel. Using well-designed primers, those 21 InDels could be amplified successfully and genotyped on the CE platform accurately and completely. The panel showed a large FST distance distinction among the ten Asian populations. Using clustering analysis, ten Asian populations were classified into three subgroups: East Asian, Southeast Asian, and South Asian subgroups. To evaluate the panel's capability in ancestry inference, a validation experiment was undertaken with 319 individuals from four geographically separated populations in China. Four Chinese populations were classified into different ancestry subgroups and 81.8% test individuals' ancestry could be inferred correctly. Our result showed that development of high ancestry informative InDels panel based on CE platform is a potential for individual ancestry inference among intracontinental populations.

Examining the additivity of peak heights in forensic DNA profiles

ABSTRACT It is routinely assumed when interpreting forensic DNA profiles that peaks of the same molecular size, whether allelic or stutter in origin, ‘stack’. That is, the height of a composite peak is approximately equal to the sum of its parts. There is strong theoretical reason to believe that this assumption should hold across the range of peak heights where fluorescent response is linear with respect to template. However, recent publications have called for empirical proof of, or directly questioned, this assumption. In this study we have examined the heights of allelic, stutter, and composite peaks, and demonstrate that peak heights are reliably predicted as the sum of their individual components. This work supports the long-held belief that peak heights ‘stack’ in an additive fashion.

First development and characterization of 27 novel microsatellite markers in the dobsonfly Neoneuromus ignobilis (Megaloptera: Corydalidae) at genome-scale level

The dobsonfly species Neoneuromus ignobilis Navas (Megaloptera: Corydalidae) is endemic to but widely distributed from eastern and southeastern Asia, being an important insect indicator for freshwater biomonitoring. At present, there is no report on the development of microsatellites of Megaloptera. Here, we developed 27 novel microsatellite markers of N. ignobilis from 850,920 candidate microsatellites with the stringent screening criteria considering the amplification success rate, the presence or absence of stutter peaks, the peak intensity, the polymorphism of the loci, the heterozygosity, and the number of alleles. The allele number of 27 microsatellite markers ranges from 3 to 12 with an average value of 6.19 per locus. The observed heterozygosity (HO) and expected heterozygosity (HE) revealed a range from 0.000 to 0.947 and 0.000 to 0.842, respectively. We constructed three panels (MP panel, most polymorphic; SS panel, most stringent strategy; ALL panel, total 27 microsatellite markers) and compared the analyses on population genetic diversity and structure. The result showed that the MP panel can significantly improve the analyses of individual assignment and genetic diversity. Accordingly, we advocate selecting the most polymorphic microsatellite marker for analyzing population genetics based on microsatellite data. The present work represents the first study on the microsatellite development of Megaloptera.

A microhap panel for kinship analysis through massively parallel sequencing technology.

It is widely recognized that microhaps are powerful markers for different forensic purposes, mainly due to their advantages of both short tandem repeats and single nucleotide polymorphisms, including multiple alleles, low mutation rate, and absence of stutter peaks. In the present study, a panel of 60 microhap loci was developed and utilized in forensic kinship analysis as a preliminary study. Genotyping of microhap was performed by massively parallel sequencing and haplotypes were directly achieved from sequence reads of 73 samples from Chinese Han population. We observed that 49 out of 60 loci have effective number of alleles greater than 3.0 and 10 out of 60 have values above 4.0, with an average value of 3.5598. The heterozygosity values were in a range from 0.5840 to 0.8546 with an average of 0.7268 and the cumulative power of exclusion value of the 60 loci is equal to 1-4.78 × 10-18 . Moreover, we demonstrated the applicability of this method by different relationship inference problems, including identification of single parent-offspring, full-sibling, and second-degree relative. The results indicated that the assembled microhap panel provided more power for relationship inference, than commonly used short tandem repeats or single nucleotide polymorphism system.

An unbalanced tri-allelic pattern at the D10S1248 locus: Characteristics, genetic basis and transmission.

To address issues related to unbalanced tri-allelic patterns, an example at the D10S1248 locus characterized by the sum of heights of alleles 13 and 15 approximately equal to allele 14 was intensively investigated. The coexistence of these three alleles was confirmed by profiling the rs2246512-D10S1248 marker using fluorescently labelled primers and allelic sequencing. Multi-tissue genotyping revealed that this pattern had chimeric characteristics, and pedigree analysis found that allele 13 or allele 14 were inherited to offspring independently of the other two alleles. These evidences suggest that the pattern should stem from a somatic mutation in early embryonic development and that peak height ratio is not an accurate indicator of mutation time point and direction. The rs2246538-D10S1248-rs2246512 marker was subsequently used to determine the mutation from allele 15 to allele 13. Notably, allele 13 with the lowest peak height was misidentified as a stutter peak when genotyped using next generation sequencing.

Testing whether stutter and low-level DNA peaks are additive

Peaks in an electropherogram could represent alleles, stutter product, or a combination of allele and stutter. Continuous probabilistic genotyping (PG) systems model the heights of peaks in an additive manner: for a shared or composite peak, PG models assume that the peak height is the sum of the allelic component and the stutter component. In this work we examine the assumption that the heights of overlapping alleles from a minor contributor and stutter peaks from a major contributor are additive.Any peak below the analytical threshold is considered unobserved; hence, in any dataset and particularly in low-template DNA profiles, some or many peaks may be unobserved or missing. Using simulation and empirical data, we show that an additive model can explain the heights of overlapping alleles from a minor contributor and stutter peaks from a major contributor as long as missing data are carefully considered. We use a naive method of imputation for the missing data which appears to perform adequately in this case. If missing data are ignored then the sum of stutter and allelic peaks is expected to be an overestimate of the average height of the composite peaks, as was observed in this study.

45. A COMPLETE SOLUTION FOR BETA-THALASSEMIA PGT TEST

Introduction Couple who are both β-thalassemia carriers usually come to PGT-M test for healthy offsprings. STR-linkage analysis is a well-known solution but Allele Drop-Out, high mutation rate and stutter peaks are still major challenges. Karyomapping is a common technique but it's costly and not be combined with PGT-A test, which may result in multiple biopsy or unaffected but aneuploidy embryos. In this study, we report two cases of β-thalassemia PGT test which uses single nucleotide polymorphisms (SNPs) for linkage analysis. By using specific primers in WGA process, the Allele Drop-out rate decreases to Materials and method Two β-thalassemia-carrier couples underwent IVF procedure, resulted in 11 embryos. Specific primers were desinged to amplify whole HBB-coding region and SNPs within 300kb flanking the HBB gene. Day 5 biopsied samples were used for whole genome amplification with designed specific primers in order to decrease ADO in HBB gene region and SNP markers, using DOPlify kit (RHS). WGA product and specific primers were used for PCR reaction to enrich HBB region and interested SNPs. Mix of WGA and enrichment PCR product was used for libraries preparation using Nextera XT DNA Library Prep Kit (Illumina) and sequenced on Miseq System. Nexus copy number was used for CNV analysis andMiseq Reporter was used for mutation detection and SNP calling. Results For the total of 11 samples, three samples resulted in aneuploidy. HBB mutation detection and SNP calling results showed no ADO in all 11 samples, with 3 affected embryos and 8 unaffected embryos. Conclusions We have successfully designed a procedure which allows to combine PGT-A and PGT-M in one single test. This test is more cost-effective and decreases turn-around time compared with other techniques. ADO rate of HBB region and SNP makers is approximately 0%, which make it easier and more reliablefor ADO confirmation among embryos in the same cycle.

Population Genetic Structure and Breeding Pattern of Cimex hemipterus (F.) (Hemiptera: Cimicidae) in Malaysia.

The surge in tropical bed bug Cimex hemipterus (Fabricius) (Hemiptera: Cimicidae) infestations has led to an increase in genomic studies. In this study, the population genetics and breeding patterns of 22 Malaysian populations were analyzed, including genetic differentiation and genetic distance. For seven microsatellite loci, the number of alleles varied from 6 to 14. The allelels per loci contrasted sharply between the overall population and within the populations. The average observed and expected heterozygosity was 0.280 and 0.828 for the overall population and 0.281 and 0.657 among the populations, respectively. Based on polymorphic information criteria, the markers with a value >0.5 were highly polymorphic. In the Hardy-Weinberg equilibrium, the loci of Ch 09ttn, Ch 01dn, and Ch 13dn of the overall population showed signs of a null allele. The stutter peaks caused no scoring errors; large allele dropouts were not detected for any loci; and a correlation imbalance was not indicated. The genetic differentiation among populations was moderate, with a coefficient of genetic differentiation (FST) of 0.144. The bed bug populations showed strong inbreeding, with highly positive coefficients of inbreeding (FIS). The molecular variation attributed to inbreeding was 83% within the populations, compared with 17% among the populations. The admixture individuals in STRUCTURE and neighbor-joining phylogenetic trees also indicated weak genetic structure in the geographical populations, suggesting moderate gene flows between populations. Thus, moderately active dispersion and human-mediated transport shaped the genetic structure of C. hemipterus populations in Malaysia.

Population Genetic Diversity and Clustering Analysis for Chinese Dongxiang Group With 30 Autosomal InDel Loci Simultaneously Analyzed.

In comparison with the most preferred genetic marker utilized in forensic science (STR), insertion/deletion analysis possesses further benefits, like absence of stutter peak, low mutation rate, and enabling mixed stain analysis. At present, a total of 169 unrelated healthy Dongxiang individuals dwelling in Dongxiang Autonomous county of Gansu province were recruited in our study to appraise the forensic usefulness of the panel including 30 autosomal diallelic genetic markers. The insertion allele frequencies were in the range of 0.1598 at HLD 111 to 0.8550 at HLD 118. The cumulative match of probability and the combined probability of exclusion were estimated based on independence of pairwise loci, with the values of 3.96 × 10-11 and 0.9886, respectively, which showed tremendous potential of this panel to be qualified for forensic personal identification in Chinese Dongxiang group. And it could also be used as a complementary tool for forensic parentage testing when combined with standard STR genetic markers. Furthermore, calculation of the DA distance and Fst values of pairwise populations, phylogenetic reconstruction, multidimensional scaling analysis, structure clustering analysis were also conducted to probe the genetic relationships between Dongxiang group and the other 30 reference populations. Results demonstrated that Dongxiang ethnic group might be genetically closer related with most Chinese populations involved in our study, especially Tibet groups, Xibe group, and several Han populations.