Accurate identification of de novo variants (DNVs) remains challenging despite advances in sequencing technologies, often requiring ad hoc filters and manual inspection. Here, we explored a purely informatic, consensus-based approach for identifying DNVs in proband-parent trios using short-read genome sequencing data. We evaluated variant calls generated by three sequence analysis pipelines-GATK HaplotypeCaller, DeepTrio, and Velsera GRAF-and examined the assumption that a requirement of consensus can serve as an effective filter for high-quality DNVs. Comparison with a highly accurate DNV set, validated previously by manual inspection and Sanger sequencing, demonstrated that consensus filtering, followed by a force-calling procedure, effectively removed false-positive calls, achieving 98.0-99.4% precision. At the same time, sensitivity of the workflow based on the previously established DNVs reached 99.4%. Validation in the HG002-3-4 Genome-in-a-Bottle trio confirmed its robustness, with precision reaching 99.2% and sensitivity up to 96.6%. We believe that this consensus approach can be widely implemented as an automated bioinformatics workflow suitable for large-scale analyses without the need for manual intervention, especially when very high precision is valued over sensitivity.

The use of compound markers has gained significant interest among forensic practitioners, due to their ability to enhance genetic marker polymorphisms by introducing new alleles. Two or more closely linked insertion/deletion (InDel) markers form a compound marker termed Multi-InDel, which offers the advantages of microhaplotype (MH) and can be genotyped using capillary electrophoresis (CE) platform. A multiplex amplification panel, including 41 Multi-InDel markers and the sex-determination locus Amelogenin, was developed and validated as an effective tool for forensic and population genetics applications.

A total of 245 Kazakh and Kyrgyz samples from China were genotyped based on the 41 Multi-InDel markers to evaluate the forensic efficacy of the panel. In addition, Multi-InDel genotyping data from 28 reference populations were collected, and population genetic analyses were performed to elucidate the genetic backgrounds of Chinese Kazakh and Kyrgyz groups.

The Multi-InDel markers demonstrated high genetic polymorphisms in Chinese Kazakh and Kyrgyz ethnic groups, indicating their suitability for forensic applications. For the two ethnic groups, the cumulative power of discrimination (CPD) values were 0.999999999999999999999999835993 and 0.999999999999999999999999717184, respectively, while the cumulative power of exclusion (CPE) values were 0.999998887418153 and 0.999999348634116, respectively. Using this Multi-InDel panel, an average of 98.82% of full sibling (FS) pairs could be distinguished from unrelated individual pairs (likelihood ratio > 1). Regarding population genetics, Chinese Kazakh and Kyrgyz groups were found to exhibit an East Asia-Europe admixed ancestry pattern, while maintaining closer genetic affinities with East Asian populations.

Despite their deleterious effects, small insertions and deletions (InDels) have received far less attention than substitutions. Here we generated isogenic CRISPR-edited human cellular models of postreplicative repair dysfunction (PRRd), including individual and combined gene edits of DNA mismatch repair (MMR) and replicative polymerases (Pol ε and Pol δ). Unique, diverse InDel mutational footprints were revealed. However, the prevailing InDel classification framework was unable to discriminate these InDel signatures from background mutagenesis and from each other. To address this, we developed an alternative InDel classification system that considers flanking sequences and informative motifs (for example, longer homopolymers), enabling unambiguous InDel classification into 89 subtypes. Through focused characterization of seven tumor types from the 100,000 Genomes Project, we uncovered 37 InDel signatures; 27 were new. In addition to unveiling previously hidden biological insights, we also developed PRRDetect-a highly specific classifier of PRRd status in tumors, with potential implications for immunotherapies.

It's important to dissect the relationship between copy number variations (CNVs) and DNA methylation, because both greatly change the dosages of genes and are responsible for diverse human cancers. Although whole genome bisulfite sequencing (WGBS) informs CNVs and DNA methylation, no study has provided a systematic benchmark for detecting CNVs from WGBS data. Herein, based on simulated and real WGBS datasets of 84.62 billion reads, we undertook 714 CNV detections to comprehensively benchmark the performance of 35 strategies, 5 alignment algorithms (bismarkbt2, bsbolt, bsmap, bwameth, and walt) wrapping with 7 CNV detection applications (BreakDancer, cn.mops, CNVkit, CNVnator, DELLY, GASV and Pindel). The results highlighted a subset of strategies that accurately called CNVs depending on numbers, lengths, precision, recall, and F1 scores of CNV detections. We found that bwameth-DELLY and bwameth-BreakDancer were the best strategies for calling deletions, and walt-CNVnator and bismarkbt2-CNVnator were the best strategies for calling duplications. These works provided investigators with useful information to accurately explore CNVs from WGBS data in humans.

The Han nationality is widely distributed in different regions, and it is one of the most populous nationalities in China. Compared with the ethnic minorities in Guangxi Zhuang Autonomous Region, there is relatively less research on Han individuals dwelled in Guangxi as a part of Chinese Han population. In this study, the genetic polymorphisms of 57 autosomal insertion/deletion (InDel) loci were explored in Hezhou Han (HZH) population. Forensic-related parameters revealed that these 57 InDel loci had high forensic validity and could be used in forensic practice application. In addition, the genetic relationships between the HZH population and 30 worldwide reference populations were explored using a variety of analytical methods, such as phylogenetic tree, principal component analysis, and genetic structure analysis. These results demonstrated that there were closer genetic relationships between the HZH and nine populations from East Asia (EAS). The prediction accuracy rates of five inter-continental cross-validation analyses for individuals from EAS was >0.9, and the prediction accuracy rates of three inter-continental cross-validation analyses for individuals from EAS, Europe, and Africa were all >0.95. In addition, 24 of the 57 InDel loci could be served as ancestral information inference loci, which could effectively distinguish individuals of EAS, Europe, and Africa. In conclusion, these InDel loci could be used not only as a good tool for individual identification and paternity testing in HZH population, but also as an auxiliary tool for ancestry information inference research.

Insertion/deletion polymorphisms, or InDels, are widely present in the human genome. They have been considered as potential markers for forensic analysis because they can be genotyped using the CE platform and compatible typing techniques used in forensic laboratories. Additionally, InDels have lower mutation rates and often short amplicon sizes, making them ideal for detecting degraded samples. However, most InDels are bi-allelic; therefore, their discrimination power is relatively low. A new set of genetic marker called multi-InDels was reported to improve InDel's informativeness. Multi-InDel markers are generally designated as microhaplotypes encompassing two or more InDels within a short distance, usually less than 200 bp. In this study, we evaluated the applicability of three previously proposed panels of multi-InDel markers, designed for Asian populations, for human identification in Brazil. We assessed all the multi-InDel markers using high-coverage whole-genome sequencing data from a census-based cohort of 1171 Brazilians residing in São Paulo, the largest Brazilian capital. The results showed that most markers are informative for Brazilian individuals since they present more than three frequent haplotypes with different sizes. However, most markers are prone to amplification/sequencing errors due to repetitive or low-complexity regions. Among the tested panels, the one from Huang et al. (2014) is the most promising for forensic use in Brazil, with a combined match probability and cumulative power of exclusion of 4.92 ×10-14 and 0.9991, respectively. Nevertheless, these values are low compared to the ones obtained with CODIS STRs (short tandem repeats) and larger SNP (single nucleotide polymorphisms) panels. Therefore, new attempts to scan the human genome for highly polymorphic multi-InDel markers are still necessary to obtain a suitable panel of multi-InDels for worldwide populations.

Somatic mutations in individual cells lead to genomic mosaicism, contributing to the intricate regulatory landscape of genetic disorders and cancers. To evaluate and refine the detection of somatic mosaicism across different technologies with personalized donor-specific assembly (DSA), we obtained tissue from the dorsolateral prefrontal cortex (DLPFC) of a post-mortem neurotypical 31-year-old individual. We sequenced bulk DLPFC tissue using Oxford Nanopore Technologies (~60X), NovaSeq (~30X), and linked-read sequencing (~28X). Additionally, we applied Cas9 capture methodology coupled with long-read sequencing (TEnCATS), targeting active transposable elements. We also isolated and amplified DNA from flow-sorted single DLPFC neurons using MALBAC, sequencing 115 of these MALBAC libraries on Nanopore and 94 on NovaSeq. We constructed a haplotype-resolved assembly with a total length of 5.77 Gb and a phase block length of 2.67 Mb (N50) to facilitate cross-platform analysis of somatic genetic variations. We observed an increase in the phasing rate from 11.6% to 38.0% between short-read and long-read technologies. By generating a catalog of phased germline SNVs, CNVs, and TEs from the assembled genome, we applied standard approaches to recall these variants across sequencing technologies. We achieved aggregated recall rates from 97.3% to 99.4% based on long-read bulk tissue data, setting an upper bound for detection limits. Moreover, utilizing haplotype-based analysis from DSA, we achieved a remarkable reduction in false positive somatic calls in bulk tissue, ranging from 14.9% to 72.4%. We developed pipelines leveraging DSA information to enhance somatic large genetic variant calling in long-read single cells. By examining somatic variation using long-reads in 115 individual neurons, we identified 468 candidate somatic heterozygous large deletions (1.5Mb - 20Mb), 137 of which intersected with short-read single-cell data. Additionally, we identified 61 putative somatic TEs (60 Alus, one LINE-1) in the single-cell data. Collectively, our analysis spans personalized assembly to single-cell somatic variant calling, providing a comprehensive ab initio ad finem approach and resource in real human tissue.

Genome-wide association study (GWAS) have identified a large number of SNPs associated with milk production traits in dairy cattle. Behind SNPs, INDELs are the second most abundant genetic polymorphisms in the genome, which may exhibit an independent association with complex traits in humans and other species. However, there are no reports on GWASs of INDELs for milk production traits in dairy cattle. In this study, using imputed sequence data, we performed INDEL-based and SNP-based GWASs for milk production traits in a Holstein cattle population. We identified 58 unique significant INDELs for one or multiple traits. The majority of these INDELs are in considerable LD with nearby significant SNPs. However, through conditional association analysis, we identified nine INDELs which showed independent associations. Genomic annotations of these INDELs indicated some novel associated genes, i.e., TRNAG-CCC, EPPK1, PPM1K, PTDSS1, and mir-10163, which were not reported in previous SNP-based GWASs. Our findings suggest that INDEL-based GWASs could be valuable complement to SNP-based GWASs for milk production traits.

Birth cohort studies involve repeated surveys of large numbers of individuals from birth and throughout their lives. They collect information useful for a wide range of life course research domains, and biological samples which can be used to derive data from an increasing collection of omic technologies. This rich source of longitudinal data, when combined with genomic data, offers the scientific community valuable insights ranging from population genetics to applications across the social sciences. Here we present quality-controlled whole exome sequencing data from three UK birth cohorts: the Avon Longitudinal Study of Parents and Children (8,436 children and 3,215 parents), the Millenium Cohort Study (7,667 children and 6,925 parents) and Born in Bradford (8,784 children and 2,875 parents). The overall objective of this coordinated effort is to make the resulting high-quality data widely accessible to the global research community in a timely manner. We describe how the datasets were generated and subjected to quality control at the sample, variant and genotype level. We then present some preliminary analyses to illustrate the quality of the datasets and probe potential sources of bias. We introduce measures of ultra-rare variant burden to the variables available for researchers working on these cohorts, and show that the exome-wide burden of deleterious protein-truncating variants, S het burden, is associated with educational attainment and cognitive test scores. The whole exome sequence data from these birth cohorts (CRAM & VCF files) are available through the European Genome-Phenome Archive, and here we provide guidance for their use.

To investigate the potential regulatory role of gene insertion or deletion (in/del) polymorphism in the occurrence of acute T cell-mediated rejection (aTCMR) after kidney transplantation.

We retrospectively analyzed the 5-year follow-up data of 133 recipients who underwent renal transplantation at the First Affiliated Hospital of Nanjing Medical University between February 1, 2010, and December 1, 2015. With target sequencing based on next-generation sequencing (NGS), tagger in/dels selection involved calculating the Hardy-Weinberg equilibrium (HWE), Minor Allele Frequency (MAF), and the linkage disequilibrium (LD) blocks. Significant in/dels associated with aTCMR were identified by intersecting the results obtained through analysis of covariance (ANCOVA) of clinical cofounders and model analysis in Rstudio using the "SNPassoc" package. Additionally, logistic models were employed to assess the associations between genotypes and the aTCMR occurrence in 5 years after surgery.

NFATc1 rs55741427 insertion was identified to be significantly associated with the post-surgery aTCMR(OR = 2.66, P < 0.001). We constructed a conclusive model containing the occurrence of delayed graft function (DGF) and the insertion polymorphism of rs55741427, showing a favorable predictive ability (AUC = 0.766) for aTCMR after surgery. Based on the receiver operating characteristic (ROC) curve, all cases were stratified into aTCMR high-risk and low-risk groups. Kaplan-Meier curves for two groups revealed that the aTCMR high-risk group exhibited a more unfavorable graft survival outcome (P = 0.0048).

Insertion mutation of rs55741427 was found to be statistically correlated with the post-surgery aTCMR during 5 years of follow-up. Our model identified DGF and insertion of rs55741427 as two crucial aTCMR-related hazards, and aTCMR high-risk group showed a worse graft prognosis.

Comprehensive characterizations of genetic diversity and demographic models of ethnolinguistically diverse Chinese populations are essential for elucidating their forensic characteristics and evolutionary past. We developed a 114-plex NGS InDel panel to genotype 114 genome-wide markers and investigated the genetic structures of Zhuang, Hui, Miao, Li, Tibetan, Yi, and Mongolian populations, encompassing five language families. This panel demonstrated robust performance, with exceptional potential for forensic individual identification and paternity testing, evidenced by the combined power of discrimination for 77 autosomal InDels (ranged from 1-3.6400 × 10-30 to 1-3.5713 × 10-32) and the combined power of exclusion (ranged from 1-2.1863 × 10-6 to 1-2.1261 × 10-7). The cumulative mean exclusion chance for 32 X-chromosomal InDels varied between 0.99996 and 0.99999 for trios and 0.99760 to 0.99898 for duos. We also analyzed genetic similarities and differences between these populations and 27 global populations, revealing distinct clusters among African, South Asian, East Asian, and European groups, with a close genetic affinity to East Asians. Notably, we identified geography-related genetic substructures: Inner Mongolia Mongolians and Gansu Huis formed a northern branch, Tibetans and Yis from Sichuan constituted a highland branch, and Guangxi Zhuangs exhibited close ties with Hainan Lis and Guangxi Miaos in the southern branch. Additionally, many InDels proved to be ancestry-informative markers for biogeographic ancestry inference. Collectively, these findings underscore the utility of the 114-plex NGS InDel panel as a complementary tool for forensic investigations and as a source of insights into the genetic architecture of ethnolinguistically distinct Chinese populations.

The genetic factors of stroke in South Asians are largely unexplored. Exome-wide sequencing and association analysis (ExWAS) in 75 K Pakistanis identified NM_000435.3(NOTCH3):c.3691 C > T, encoding the missense amino acid substitution p.Arg1231Cys, enriched in South Asians (alternate allele frequency = 0.58% compared to 0.019% in Western Europeans), and associated with subcortical hemorrhagic stroke [odds ratio (OR) = 3.39, 95% confidence interval (CI) = [2.26, 5.10], p = 3.87 × 10-9), and all strokes (OR [CI] = 2.30 [1.77, 3.01], p = 7.79 × 10-10). NOTCH3 p.Arg231Cys was strongly associated with white matter hyperintensity on MRI in United Kingdom Biobank (UKB) participants (effect [95% CI] in SD units = 1.1 [0.61, 1.5], p = 3.0 × 10-6). The variant is attributable for approximately 2.0% of hemorrhagic strokes and 1.1% of all strokes in South Asians. These findings highlight the value of diversity in genetic studies and have major implications for genomic medicine and therapeutic development in South Asian populations.

Insertions and deletions constitute the second most important source of natural genomic variation. Insertions and deletions make up to 25% of genomic variants in humans and are involved in complex evolutionary processes including genomic rearrangements, adaptation, and speciation. Recent advances in long-read sequencing technologies allow detailed inference of insertions and deletion variation in species and populations. Yet, despite their importance, evolutionary studies have traditionally ignored or mishandled insertions and deletions due to a lack of comprehensive methodologies and statistical models of insertions and deletion dynamics. Here, we discuss methods for describing insertions and deletion variation and modeling insertions and deletions over evolutionary time. We provide practical advice for tackling insertions and deletions in genomic sequences and illustrate our discussion with examples of insertions and deletion-induced effects in human and other natural populations and their contribution to evolutionary processes. We outline promising directions for future developments in statistical methodologies that would allow researchers to analyze insertions and deletion variation and their effects in large genomic data sets and to incorporate insertions and deletions in evolutionary inference.

Childhood cancer survivorship studies generate comprehensive datasets comprising demographic, diagnosis, treatment, outcome, and genomic data from survivors. To broadly share this data, we created the St. Jude Survivorship Portal (https://survivorship.stjude.cloud), the first data portal for sharing, analyzing, and visualizing pediatric cancer survivorship data. More than 1,600 phenotypic variables and 400 million genetic variants from more than 7,700 childhood cancer survivors can be explored on this free, open-access portal. Summary statistics of variables are computed on-the-fly and visualized through interactive and customizable charts. Survivor cohorts can be customized and/or divided into groups for comparative analysis. Users can also seamlessly perform cumulative incidence and regression analyses on the stored survivorship data. Using the portal, we explored the ototoxic effects of platinum-based chemotherapy, uncovered a novel association between mental health, age, and limb amputation, and discovered a novel haplotype in MAGI3 strongly associated with cardiomyopathy specifically in survivors of African ancestry. Significance: The St. Jude Survivorship Portal is the first data portal designed to share and explore clinical and genetic data from childhood cancer survivors. The portal provides both open- and controlled-access features and will fulfill a wide range of data sharing needs of the survivorship research community and beyond. See co-corresponding author Xin Zhou discuss this research article, published simultaneously at the AACR Annual Meeting 2024: https://vimeo.com/932617204/7d99fa4958.

The paper presents a review of mechanistic modelling studies of DNA damage and DNA repair, and consequences to follow in mammalian cell nucleus. We hypothesize DNA deletions are consequences of repair of double strand breaks leading to the modifications of genome that play crucial role in long term development of genetic inheritance and diseases. The aim of the paper is to review formation mechanisms underlying naturally occurring DNA deletions in the human genome and their potential relevance for bridging the gap between induced DNA double strand breaks and deletions in damaged human genome from endogenous and exogenous events. The model of the cell nucleus presented enables simulation of DNA damage at molecular level identifying the spectrum of damage induced in all chromosomal territories and loops. Our mechanistic modelling of DNA repair for double stand breaks (DSB), single strand breaks (SSB) and base damage (BD), shows the complexity of DNA damage is responsible for the longer repair times and the reason for the biphasic feature of mammalian cells repair curves. In the absence of experimentally determined data, the mechanistic model of repair predicts the in vivo rate constants for the proteins involved in the repair of DSB, SSB, and of BD.

Breast cancer imposes a significant burden globally. While the survival rate is steadily improving, much remains to be elucidated. This observational, single time point, multiomic study utilizing genomics, proteomics, targeted and untargeted metabolomics, and metagenomics in a breast cancer survivor (BCS) and age-matched healthy control cohort (N = 100) provides deep molecular phenotyping of breast cancer survivors. In this study, the BCS cohort had significantly higher polygenic risk scores for breast cancer than the control group. Carnitine and hexanoyl carnitine were significantly different. Several bile acid and fatty acid metabolites were significantly dissimilar, most notably the Omega-3 Index (O3I) (significantly lower in BCS). Proteomic and metagenomic analyses identified group and pathway differences, which warrant further investigation. The database built from this study contributes a wealth of data on breast cancer survivorship where there has been a paucity, affording the ability to identify patterns and novel insights that can drive new hypotheses and inform future research. Expansion of this database in the treatment-naïve, newly diagnosed, controlling for treatment confounders, and through the disease progression, can be leveraged to profile and contextualize breast cancer and breast cancer survivorship, potentially leading to the development of new strategies to combat this disease and improve the quality of life for its victims.

The use of genetic markers, specifically Short Tandem Repeats (STRs), has been a valuable tool for identifying persons of interest. However, the ability to analyze additional markers including Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletion (INDELs) polymorphisms allows laboratories to explore other investigative leads. INDELs were chosen in this study because large panels can be differentiated by size, allowing them to be genotyped by capillary electrophoresis. Moreover, these markers do not produce stutter and are smaller in size than STRs, facilitating the recovery of genetic information from degraded samples. The INDEL Ancestry Informative Markers (AIMs) in this study were selected from the 1000 Genomes Project based on a fixation index (FST) greater than 0.50, high allele frequency divergence, and genetic distance. A total of 25 INDEL-AIMs were optimized and validated according to SWGDAM guidelines in a five-dye multiplex. To validate the panel, genotyping was performed on 155 unrelated individuals from four ancestral groups (Caucasian, African, Hispanic, and East Asian). Bayesian clustering and principal component analysis (PCA) were performed revealing clear separation among three groups, with some observed overlap within the Hispanic group. Additionally, the PCA results were compared against a training set of 793 samples from the 1000 Genomes Project, demonstrating consistent results. Validation studies showed the assay to be reproducible, tolerant to common inhibitors, robust with challenging casework type samples, and sensitive down to 125 pg. In conclusion, our results demonstrated the robustness and effectiveness of a 25 loci INDEL system for ancestry inference of four ancestries commonly found in the United States.

Insertion/Deletion (InDel) polymorphisms, characterized by their smaller amplicons, reduced mutation rates, and compatibility with the prevalent capillary electrophoresis (CE) platforms in forensic laboratories, significantly contribute to the advancement and application of genetic analysis. Guizhou province in China serves as an important region for investigating the genetic structure, ethnic group origins, and human evolution. However, DNA data and the sampling of present-day populations are lacking, especially about the InDel markers. Here, we reported data on 47 autosomal InDels from 592 individuals from four populations in Guizhou (Han, Dong, Yi, and Chuanqing). Genotyping was performed with the AGCU InDel 50 kit to evaluate their utility for forensic purposes and to explore the population genetic structure. Our findings showed no significant deviations from Hardy-Weinberg and linkage equilibriums. The combined power of discrimination (CPD) and the combined power of exclusion (CPE) for each population demonstrated that the kit could be applied to forensic individual identification and was an effective supplement for parentage testing. Genetic structure analyses, including principal component analysis, multidimensional scaling, genetic distance calculation, STRUCTURE, and phylogenetic analysis, highlighted that the genetic proximity of the studied populations correlates with linguistic, geographical, and cultural factors. The observed genetic variances within four research populations were less pronounced than those discerned between populations across different regions. Notably, the Guizhou Han, Dong, and Chuanqing populations showed closer genetic affiliations with linguistically similar groups than the Guizhou Yi. These results underscore the potential of InDel markers in forensic science and provide insights into the genetic landscape and human evolution in multi-ethnic regions like Guizhou.

InDel markers show promise for forensic individual identification and parentage testing via the AGCU InDel 50 kit.Genetic analysis of Guizhou populations reveals correlations with linguistic, geographical, and cultural factors.Guizhou Han, Dong, and Chuanqing populations showed closer genetic affiliations with linguistically similar groups than the Guizhou Yi.

In recent years, the insertion/deletion (InDel) polymorphism has become a preferred genetic marker in forensic genetics due to its low mutation rates and small amplicon sizes. In this study, a 36-InDelplex identification panel, consisting of autosomal 34 InDel loci, 1 Y InDel locus, and amelogenin, was developed, and gene frequencies in the Turkish population were determined. The loci of the InDel panel with global minimum allele frequencies (MAF) ≥ 0.4 were selected from the 1000 Genomes Project Phase 3 data. The amplicon sizes of the loci were designed in the range of 69-252 bp. In the validation study of the developed panel, analysis threshold, dynamic range, sensitivity, stochastic threshold, inhibitor tolerance, and reproducibility parameters were studied by following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The sensitivity studies indicated that complete and reliable InDel profiles could be obtained with 0.25 ng of DNA. A population study was evaluated using 250 samples from Turkey. The mean observed heterozygosity ratio (Ho) of all loci was 0.48. The combined discrimination power (CPD) is 0.999999999990867 and the combined exclusion probability (CPE) was 0.9930. The population comparison was also made using Turkish and the five major populations from the 1000 Genomes Phase 3 populations' data (Africa, Europe, East Asia, South Asia, and America). In conclusion, the results showed that the 36-InDelplex panel is a reliable, sensitive, and accurate system that is suitable for human identification and population genetics purposes.

Accurate estimation of population allele frequency (AF) is crucial for gene discovery and genetic diagnostics. However, determining AF for frameshift-inducing small insertions and deletions (indels) faces challenges due to discrepancies in mapping and variant calling methods. Here, we propose an innovative approach to assess indel AF. We developed CRAFTS-indels (Calculating Regional Allele Frequency Targeting Small indels), an algorithm that combines AF of distinct indels within a given region and provides "regional AF" (rAF). We tested and validated CRAFTS-indels using three independent datasets: gnomAD v2 (n=125,748 samples), an internal dataset (IGM; n=39,367), and the UK BioBank (UKBB; n=469,835). By comparing rAF against standard AF, we identified rare indels with rAF exceeding standard AF (sAF≤10-4 and rAF>10-4) as "rAF-hi" indels. Notably, a high percentage of rare indels were "rAF-hi", with a higher proportion in gnomAD v2 (11-20%) and IGM (11-22%) compared to the UKBB (5-9% depending on the CRAFTS-indels' parameters). Analysis of the overlap of regions based on their rAF with low complexity regions and with ClinVar classification supported the pertinence of rAF. Using the internal dataset, we illustrated the utility of CRAFTS-indel in the analysis of de novo variants and the potential negative impact of rAF-hi indels in gene discovery. In summary, annotation of indels with cohort specific rAF can be used to handle some of the limitations of current annotation pipelines and facilitate detection of novel gene disease associations. CRAFTS-indels offers a user-friendly approach to providing rAF annotation. It can be integrated into public databases such as gnomAD, UKBB and used by ClinVar to revise indel classifications.

DNA analysis of forensic case samples relies on short tandem repeats (STRs), a key component of the combined DNA index system (CODIS) used to identify individuals. However, limitations arise when dealing with challenging samples, prompting the exploration of alternative markers such as single nucleotide polymorphisms (SNPs) and insertion/deletion (INDELs) polymorphisms. Unlike SNPs, INDELs can be differentiated easily by size, making them compatible with electrophoresis methods. It is possible to design small INDEL amplicons (<200 bp) to enhance recovery from degraded samples. To this end, a set of INDEL Human Identification Markers (HID) was curated from the 1000 Genomes Project, employing criteria including a fixation index (FST) ≤ 0.06, minor allele frequency (MAF) >0.2, and high allele frequency divergence. A panel of 33 INDEL-HIDs was optimized and validated following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, utilizing a five-dye multiplex electrophoresis system. A small sample set (n = 79 unrelated individuals) was genotyped to assess the assay's performance. The validation studies exhibited reproducibility, inhibition tolerance, ability to detect a two-person mixture from a 4:1 to 1:6 ratio, robustness with challenging samples, and sensitivity down to 125 pg of DNA. In summary, the 33-loci INDEL-HID panel exhibited robust recovery with low-template and degraded samples and proved effective for individualization within a small sample set.

Accurate identification of germline de novo variants (DNVs) remains a challenging problem despite rapid advances in sequencing technologies as well as methods for the analysis of the data they generate, with putative solutions often involving ad hoc filters and visual inspection of identified variants. Here, we present a purely informatic method for the identification of DNVs by analyzing short-read genome sequencing data from proband-parent trios. Our method evaluates variant calls generated by three genome sequence analysis pipelines utilizing different algorithms-GATK HaplotypeCaller, DeepTrio and Velsera GRAF-exploring the assumption that a requirement of consensus can serve as an effective filter for high-quality DNVs. We assessed the efficacy of our method by testing DNVs identified using a previously established, highly accurate classification procedure that partially relied on manual inspection and used Sanger sequencing to validate a DNV subset comprising less confident calls. The results show that our method is highly precise and that applying a force-calling procedure to putative variants further removes false-positive calls, increasing precision of the workflow to 99.6%. Our method also identified novel DNVs, 87% of which were validated, indicating it offers a higher recall rate without compromising accuracy. We have implemented this method as an automated bioinformatics workflow suitable for large-scale analyses without need for manual intervention.

Insertion/deletion polymorphisms (InDels) are a category of highly prevalent markers in the human genome, characterized by their distinctive attributes, including short amplicon sizes and low mutation rates, which have shown great potential in forensic applications. Multi-allelic InDel and multi-InDel markers, collectively abbreviated as MM-InDels, were developed to enhance polymorphism by the introduction of novel alleles. Nevertheless, the relatively low mutation rates of InDels, coupled with the founder effect, result in distinct allele frequency distributions among populations. The divergent characteristics of InDels in different populations also pose challenges to the establishment of universally efficient InDel multiplex assays. To enhance the system efficiency of the InDel assay and its applicability across diverse populations, 39 MM-InDels with high polymorphism in five different ancestry superpopulations were selected from the 1000 Genomes Project dataset and combined with an amelogenin gender marker to construct a multiplex assay (named MMIDplex). The combined power of discrimination and the cumulative probability of exclusion of 39 MM-InDels were 1 - 1.3 × 10-23 and 1 - 9.83 × 10-6 in the Chinese Han population, and larger than 1-10-19 and 1-10-4 in the reference populations, relatively. These results demonstrate that the MMIDplex assay has the potential to obtain sufficient power for individual identification and paternity test in global populations.

Deletion/insertion polymorphisms (DIPs), a novel class of biomarker, have been widely utilized in forensic areas for individual identification, paternity tests, and ancestral origin inference due to its applicability to degraded samples and low mutation rates. Despite the availability of a well-established commercial kit, the Investigator® DIPplex kit (Qiagen), certain loci exhibit limited levels of polymorphisms in East Asian populations, particularly in Chinese populations.

This dissertation seeks to undertake a comprehensive evaluation about the forensic efficiency of a self-developed multiplex amplification system in high-altitude adaptive ethnic groups of China. Healthy unrelated Tibetan individuals residing in Tibet Autonomous Region and Qinghai Province were genotyped using previously reported 43 deletion/insertion polymorphism loci. Forensic statistical analyses including allele frequencies and forensic parameters were conducted in the two Tibetan groups, and the genetic relatedness of the studied groups with reference populations from the 1000 Genomes Project Phase 3 were investigated.

Forensic statistical results showed that the polymorphism information content values of the 43 deletion/insertion polymorphism loci in the two Tibetan groups exceeded 0.35. Moreover, the combined power of discrimination using the 43 deletion/insertion polymorphism loci was calculated to be 0.9999999999999999984 in the Qinghai Tibetan group and 0.9999999999999999921 in the Tibet Tibetan group. The cumulative power of exclusion using the 43 deletion/insertion polymorphism loci was calculated to be 0.999782512 in the Qinghai Tibetan group and 0.999886205 in the Tibet Tibetan group. Analysis of population genetics demonstrated that the two studied Tibetan groups shared close genetic relationships with East Asia populations.

The set of 43 deletion/insertion polymorphism loci exhibited remarkable forensic efficacy, rendering it a promising tool for forensic practice. Population genetic analyses indicated that the two Tibetan groups had closer genetic affinities to East Asian populations.

The Mexico City Prospective Study is a prospective cohort of more than 150,000 adults recruited two decades ago from the urban districts of Coyoacán and Iztapalapa in Mexico City1. Here we generated genotype and exome-sequencing data for all individuals and whole-genome sequencing data for 9,950 selected individuals. We describe high levels of relatedness and substantial heterogeneity in ancestry composition across individuals. Most sequenced individuals had admixed Indigenous American, European and African ancestry, with extensive admixture from Indigenous populations in central, southern and southeastern Mexico. Indigenous Mexican segments of the genome had lower levels of coding variation but an excess of homozygous loss-of-function variants compared with segments of African and European origin. We estimated ancestry-specific allele frequencies at 142 million genomic variants, with an effective sample size of 91,856 for Indigenous Mexican ancestry at exome variants, all available through a public browser. Using whole-genome sequencing, we developed an imputation reference panel that outperforms existing panels at common variants in individuals with high proportions of central, southern and southeastern Indigenous Mexican ancestry. Our work illustrates the value of genetic studies in diverse populations and provides foundational imputation and allele frequency resources for future genetic studies in Mexico and in the United States, where the Hispanic/Latino population is predominantly of Mexican descent.

Milk thistle (Silybum marianum) belongs to the Asteraceae family and is a medicinal plant native to the Mediterranean Basin. Silymarin in achene is a widely used herbal product for chronic liver disease. There is growing interest in natural medicine using milk thistle in Korea, but the raw material completely relies on imports. Despite its economic importance, phenotypic evaluations of native resources of milk thistle in Korea have not been carried out. In addition, genomic research and molecular marker development are very limited in milk thistle. In this study, we evaluated 220 milk thistle resources consisting of 172 accessions collected from the domestic market, and 48 accessions isolated from 6 accessions distributed by the National Agrobiodiversity Center in Korea. Six plant characteristics (height, seed weight, number of flowers, seed weight per flower, spine length, and color at harvest) were measured, and six samples (M01-M06) were selected to represent the genetic diversity of the population for genomic research. To develop PCR-based and co-dominant insertion/deletion (InDel) markers, we performed genome-wide InDel detection by comparing the whole-genome resequencing data of the six selected accessions with the reference genome sequence (GCA_001541825). As a result, 177 InDel markers with high distinguishability and reproducibility were selected from the 30,845 InDel variants. Unknowingly imported alien plant resources could easily be genetically mixed, and jeopardized seed purity can cause continuous difficulties in the development of high value-added agricultural platforms utilizing natural products. The selected plant materials and 177 validated InDel markers developed via whole-genome resequencing analysis could be valuable resources for breeding, conservation, and ecological studies of natives to Korea, along with acceleration of Silybum marianum industrialization.

Citrus is a source of nutritional and medicinal advantages, cultivated worldwide with major groups of sweet oranges, mandarins, grapefruits, kumquats, lemons and limes. Pakistan produces all major citrus groups with mandarin (Citrus reticulata) being the prominent group that includes local commercial cultivars Feutral's Early, Dancy, Honey, and Kinnow. The present study designed to understand the genetic architecture of this unique variety of Citrus reticulata 'Kinnow.' The whole-genome resequencing and variant calling was performed to map the genomic variability that might be responsible for its particular characteristics like taste, seedlessness, juice content, thickness of peel, and shelf-life. A total of 139,436,350 raw sequence reads were generated with 20.9 Gb data in Fastq format having 98% effectiveness and 0.2% base call error rate. Overall, 3,503,033 SNPs, 176,949 MNPs, 323,287 INS, and 333,083 DEL were identified using the GATK4 variant calling pipeline against Citrus clementina. Furthermore, g:Profiler was applied for annotating the newly found variants, harbor genes/transcripts and their involved pathways. A total of 73,864 transcripts harbors 4,336,352 variants, most of the observed variants were predicted in non-coding regions and 1009 transcripts were found well annotated by different databases. Out of total aforementioned transcripts, 588 involved in biological processes, 234 in molecular functions and 167 transcripts in cellular components. In a nutshell, 18,153 high impact variants and 216 genic variants found in the current study, which may be used after its functional validation for marker-assisted breeding programs of "Kinnow" to propagate its valued traits for the improvement of contemporary citrus varieties in the region.

Yunnan is one of the main residences of the Zhuang group which is one of the 55 ethnic minorities in China. At present, there are relatively few researches on population genetics and forensic science of the Yunnan Zhuang group. Therefore, this study used a self-constructed panel containing 41 multi-InDel markers to analyze the genetic polymorphisms of 173 individuals from Yunnan Zhuang group. The results indicated that these 41 multi-InDels in Yunnan Zhuang group were highly polymorphic markers expect for three markers. The cumulative match probability and combined exclusion probability values of the 40 multi-InDels (MI38 marker was excluded) were 8.0671E-26 and 0.9999995959, respectively. In addition, population genetic analyses were performed on genotyping data of 41 multi-InDel markers among the Yunnan Zhuang and 26 reference populations, revealing that the Yunnan Zhuang group was genetically close to the five populations in East Asia. According to the STRUCTURE analysis, the Yunnan Zhuang group presented similar ancestral compositions to the five populations from East Asia, and when the K value was three, the five intercontinental populations showed their different genetic structures. In conclusion, the 41 multi-InDel markers could be used as an effective tool for individual identification and paternity testing of the Zhuang group in Yunnan province, as well as for their ancestry information inference studies.

Insertion and deletion mutations (indels) are important mechanisms of generating protein diversity. Indels in coding sequences are under considerable selective pressure to maintain reading frames and to preserve protein function, but once generated, indels provide raw material for the acquisition of new protein properties and functions. We reported recently that coding sequence insertions in the Candida albicans NDU1 protein, a mitochondrial protein involved in the assembly of the NADH:ubiquinone oxidoreductase are imperative for respiration, biofilm formation and pathogenesis. NDU1 inserts are specific to CTG-clade fungi, absent in human ortholog and successfully harnessed as drug targets. Here, we present the first comprehensive report investigating indels and clade-defining insertions (CDIs) in fungal proteomes. We investigated 80 ascomycete proteomes encompassing CTG clade species, the Saccharomycetaceae family, the Aspergillaceae family and the Herpotrichiellaceae (black yeasts) family. We identified over 30,000 insertions, 4,000 CDIs and 2,500 clade-defining deletions (CDDs). Insert sizes range from 1 to over 1,000 residues in length, while maximum deletion length is 19 residues. Inserts are strikingly over-represented in protein kinases, and excluded from structural domains and transmembrane segments. Inserts are predicted to be highly disordered. The amino acid compositions of the inserts are highly depleted in hydrophobic residues and enriched in polar residues. An indel in the Saccharomyces cerevisiae Sth1 protein, the catalytic subunit of the RSC (Remodel the Structure of Chromatin) complex is predicted to be disordered until it forms a ß-strand upon interaction. This interaction performs a vital role in RSC-mediated transcriptional regulation, thereby expanding protein function.

Dyslipidemias are risk factors in diseases of significant importance to public health, such as atherosclerosis, a condition that contributes to the development of cardiovascular disease. Unhealthy lifestyles, the pre-existence of diseases, and the accumulation of genetic variants in some loci contribute to the development of dyslipidemia. The genetic causality behind these diseases has been studied primarily on populations with extensive European ancestry. Only some studies have explored this topic in Costa Rica, and none have focused on identifying variants that can alter blood lipid levels and quantifying their frequency. To fill this gap, this study focused on identifying variants in 69 genes involved in lipid metabolism using genomes from two studies in Costa Rica. We contrasted the allelic frequencies with those of groups reported in the 1000 Genomes Project and gnomAD and identified potential variants that could influence the development of dyslipidemias. In total, we detected 2,600 variants in the evaluated regions. However, after various filtering steps, we obtained 18 variants that have the potential to alter the function of 16 genes, nine variants have pharmacogenomic or protective implications, eight have high risk in Variant Effect Predictor, and eight were found in other Latin American genetic studies of lipid alterations and the development of dyslipidemia. Some of these variants have been linked to changes in blood lipid levels in other global studies and databases. In future studies, we propose to confirm at least 40 variants of interest from 23 genes in a larger cohort from Costa Rica and Latin American populations to determine their relevance regarding the genetic burden for dyslipidemia. Additionally, more complex studies should arise that include diverse clinical, environmental, and genetic data from patients and controls and functional validation of the variants.

The insertion/deletion (InDel) polymorphism has promising applications in forensic DNA analysis. However, the insufficient forensic efficiencies of the present InDel-based systems restrict their applications in parentage testing, due to the lower genetic polymorphism of the biallelic InDel locus and the limited number of InDel loci in a multiplex amplification system. Here, we introduced an in-house developed system which contained 41 polymorphic Multi-InDel markers (equivalent to 82 InDels in total), to serve as an efficient and reliable tool for different forensic applications in the Manchu and Mongolian groups. We demonstrated that the new system exhibited potential efficiencies for personal identification, parentage testing, two-person DNA mixture interpretation and ancestry inference of intercontinental populations. Meanwhile, we explored the genetic backgrounds of the Manchu and Mongolian groups by conducting a series of population genetic analyses. We showed that the Manchu and Mongolian groups shared closer genetic relationships with East Asian populations, especially Han Chinese populations in northern China. Moreover, more similar genetic compositions were detected between the Manchu group and the northern Han populations in this study, suggesting that the Manchu group had higher genetic affinities with northern Han populations than the Mongolian group. Overall. this study provided the necessary evidence that these Multi-InDel genetic markers could play an important role in forensic applications.

InDel (Insertions/deletion) markers have been used as an alternative, or as a complementary marker system, to STR markers in human identification due to their advantages such as low mutation rates, no stutter, and potential small amplicon sizes. In forensic sciences, sex chromosomes are widely used in forensic genetics for specific cases. For example, the relationship between father and daughter can be determined by using X-InDels. In this study, we developed a novel 22 X-InDel multiplex system that was identified by two separate assays with fluorescence amplification and capillary electrophoresis detection technology. We chose 22 X-InDel markers based on the following criteria: mean heterozygosity over 30% in Europeans; minimum of 250 Kb differences between each InDel loci; and an amplicon length that was less than 300 bp. We performed an optimization and validation study of 22 X-InDel systems under the following parameters: analytical threshold, sensitivity, precision and accuracy, stochastic threshold, repeatability, and reproducibility. We evaluated the allele frequency of this multiplex system in the Turkish population, and then the population comparisons were carried out on data from 1000 Genome populations (Europe, Africa, America, South Asia, and East Asia). The sensitivity test showed a complete genotyping profile with DNA concentrations as low as 0.5 ng. The heterozygosity ratio of 22 X-InDel loci was determined as 0.4690 and the discrimination power was defined as 0.99. The results show that the new 22 X-InDel multiplex system provides high polymorphism information, and it is a reproducible, accurate, sensitive, and robust system that could be used as an additional tool for kinship testing.

Schizophrenia is a psychiatric syndrome that affects approximately 1 % of the world population and is among the top 10 reasons for disability. In this case-control study, we investigated the association between 15 insertion/deletion (Indel) polymorphisms and schizophrenia risk using pooled samples. In the present case-control study, 361 individuals with schizophrenia and 360 healthy individuals were included in the study. We examined the insertion/deletion polymorphisms in APOB, ADRA2B, PDCD6IP, LRPAP1, TLR2, DHFR, VEGF, HLA-G, TPA, DBH, UCP2, FADS2, MDM2, TP53 and SLC6A4 genes. Our results revealed that the Del allele of the HLA-G 14bp Indel polymorphism increased the risk of schizophrenia (OR=1.23, 95 % CI=1.01-1.52, p=0.045) and the Alu- allele of the TPA Alu+/Alu- polymorphism negatively associated with the schizophrenia risk (OR=0.67, 95 % CI=0.54-0.82, p<0.001).

In the present study, the re-sequencing of our previous whole-genome sequencing (WGS) for selected individuals of Dazu-black goat (DBG) and Inner-Mongolia Cashmere goat (IMCG) breeds was used to detect and compare the differentiation in Indels depending on the reference genome of goat. Then, three selected candidate Indels rs668795676, rs657996810, and rs669452874 of the three genes SUFU, SYCP2L and GLIPR1L1, respectively, have been chosen, based on the results of prior GWAS across the genome, and examined for their association with body weight and dimensions (body height, body length, heart girth, chest width, cannon circumference, and chest depth) by kompetitive allele specific PCR assay for 342 goats from the three studied goat breeds (DBG, n = 203; ♂99, ♀104), IMCG (n = 65; 15♂, 50♀), and Hechuan white goat (HWG, n = 74; 34♂, 40♀) breeds. The analysis of 192.747 Gb WGS revealed an average 334,151 Indels in the whole genome of DBG and IMCG breeds. Chromosome 1 had a maximum number of mutations (Indels) of 58,497 and 55,527 for IMCG and DBG, respectively, while chromosome 25 had the least number of mutations of 15,680 and 16,103 for IMCG and DBG, respectively. The majority of Indels were either Ins or Del of short fragments of 1-5 bp, which covered 79.06 and 71.78% of the total number of Indels mutations in IMCG and DBG, respectively. Comparing the differences of Indels between the studied goat breeds revealed 100 and 110 unique Indels for IMCG and DBG, respectively. The Indels loci in the intron region were unique for both studied goat breeds which were related to 30 and 38 candidate genes in IMCG and DBG, respectively, including SUFU, SYCP2L, and GLIPR1L1 genes. Concerning rs669452874 locus, body height and body length of Del/Del genotype in DBG were significantly higher (P < 0.05) than that of Ins/Del genotype, while body height and body length of Del/Del genotype in IMCG were significantly higher (P < 0.01) than those of Ins/Ins genotype, whereas body height and body length and heart girth of Del/Del genotype in HWG were significantly higher (P < 0.01) than those of the Ins/Del and Ins/Ins genotypes. Thus, Del/Del genotype of rs669452874 locus can be used as a candidate molecular marker related to the body dimensions in the studied goat breeds.