Genomic View Research Articles

Amplicon-based long-read sequencing is useful for confirming zygosity of DSG2 variants associated with Japanese ARVC

Abstract Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy mainly caused by desmosomal gene variants. Previously, we reported that DSG2 variants were the most common in 153 Japanese ARVC probands (DSG2 multiple, n=62 and single, n=4; PKP2, n=28; others, n=7). Among the multiple DSG2 variant carriers, 22 probands were compound heterozygous carriers, and 25 were homozygous, which were confirmed by genetic testing for family members (Figure A). On the other hand, 88% of their family members with single DSG2 variant carriers were not diagnosed with ARVC, which meant that recessive DSG2 variants were the major cause of ARVC in Japan. However, zygosities in remaining 15 probands with multiple DSG2 variants were unknown due to the lack of genome data from their parents. Purpose This study aimed to clarify the zygosities of multiple DSG2 variants using amplicon-based long-read sequencing. Methods Genomic DNA was extracted from whole blood using standard protocols. The range containing multiple variants was amplified by long-range PCR. Sequencing libraries were prepared using ONT Ligation Sequencing Kit. Each library was loaded onto a flow cell for sequencing on ONT GridION Mk1 running MinKNOW control software. FASTQ files were aligned to hg38 using minimap2. Variants were called using Clair3 and phased using WhatsHap. Integrative Genomics Viewer (IGV) was used for visualization of the phased variants. Results We successfully phased all the multiple DSG2 variants found in the 15 proband. A typical result of amplicon-based LRS was shown in Figure B. It was IGV screen shot of reads at DSG2 containing c.874C>T (p.R292C) and c.1481A>C (p.D494A). The reads colored in red belonged to one haplotype, while the ones in blue belonged to the other. Each of the two variants was on different color reads, indicating they were compound heterozygous variants. Using LRS, we confirmed all the 15 probands had compound heterozygous variants in DSG2. Overall, the 62 probands (40.5%) with DSG2 variants were recessive variant carriers (Figure A). Conclusions Japanese ARVC was mainly caused by autosomal recessive variants in DSG2. Clarifying the zygosity of causative variants is crucial for the diagnosis in recessive diseases including Japanese ARVC. Amplicon-based LRS was helpful for phasing the multiple DSG2 variants directly without genetic testing of the parents.Figure AFigure B

Copy Number Analysis of Whole Exome Sequencing Data

Abstract Background Genetic alterations implicated in constitutional disorders range in size from single nucleotide substitutions to losses and gains of millions of bases. Whole exome sequencing (WES) captures many clinically relevant variants, but an analytic pipeline optimized for detection of small substitutions, insertions, and deletions may miss pathogenic copy number variants (CNVs). Historically, our laboratory performed separate cytogenetic tests to evaluate for larger genomic imbalances. Addition of copy number analysis to our WES pipeline could improve the assay’s diagnostic yield. This study describes part of our strategy to validate copy number analysis of our constitutional whole exome sequencing data. Methods We identified 18 CNV specimens that underwent both chromosomal microarray analysis (CMA) and WES between 2017 and 2023. Variants included 13 deletions (size range 14Kb-2Mb) and 5 duplications (size range 325Kb-10Mb). The standard method of constitutional copy number analysis in our clinical laboratory is CMA using the Affymetrix Cytoscan HD Array and analysis software. CNVs identified on CMA were reviewed with reference to the Database of Genomic Variants and multiple academic hospital databases. WES was performed on an Illumina sequencing system following capture-based library enrichment with the Integrated DNA Technologies xGen Exome Research, CNV Backbone, Human mtDNA Research, and Human ID Research Panel probes. CNVs identified on WES were called using the Broad Institute’s Genomic Analysis Toolkit (GATK) with a quality score cutoff of 120. Whole exome sequencing data was visualized on the Integrative Genomics Viewer. Results 14 out of 18 CNVs reported on CMA were also detected on WES with a quality score at or above 120 (78%). This includes 10 out of 13 deletions (77%) and 3 out of 5 duplications (60%). For both deletions and duplications, the size ranges of detected variants at or above the cutoff score and undetected variants overlapped. One duplication reported on CMA was detected on WES with a quality score below 120. Visualization of whole exome sequencing data showed variation in probe density that may have affected CNV calling. Conclusions The majority of CNVs reported after CMA (14/18, 78%) were also detected on WES data using a GATK quality score cutoff of 120. The data does not suggest an association between CNV size and detection by WES, although interpretation is limited by the small number of cases. Data visualization may be required to identify some CNVs. Future steps to characterize the limitations of copy number analysis of WES data include assessments of variance in probe density and read depth across the exome. Copy number analysis of WES data from cases with normal CMA results will also be performed for assay validation.

The first Chromosomal-level genome assembly of Sageretia thea using Nanopore long reads and Pore-C technology

Sageretia thea, a notable species within the mock buckthorn genus, is recognized for its intriguing biogeographical distribution and diverse medicinal properties. Despite this significance, genomic studies on S. thea are still in the nascent stages. We present the first chromosome-level genome assembly of S. thea that was generated using a combination of Oxford Nanopore long-read and Illumina short-read sequencing technologies complemented by Pore-C chromatin conformation capture. The genome assembly had a size of 197.8 Mb with 12 chromosomal scaffolds and a scaffold N50 length of 15.9 Mb. A total of 25,434 protein-coding genes were identified and functionally annotated, and the gene model indicated 96.5% complete eukaryotic BUSCOs. Additionally, orthologous gene profiling and synteny analysis were performed to elucidate the evolutionary relationships within the Rhamnaceae family and Rosales. This high-quality chromosomal genome is the first genomic view of S. thea, which will serve as the basis for future studies on its biological and medicinal properties, and evolutionary history.

Hijackers, hitchhikers, or co-drivers? The mysteries of mobilizable genetic elements.

Mobile genetic elements shape microbial gene repertoires and populations. Recent results reveal that many, possibly most, microbial mobile genetic elements require helpers to transfer between genomes, which we refer to as Hitcher Genetic Elements (hitchers or HGEs). They may be a large fraction of pathogenicity and resistance genomic islands, whose mechanisms of transfer have remained enigmatic for decades. Together with their helper elements and their bacterial hosts, hitchers form tripartite networks of interactions that evolve rapidly within a parasitism-mutualism continuum. In this emerging view of microbial genomes as communities of mobile genetic elements many questions arise. Which elements are being moved, by whom, and how? How often are hitchers costly hyper-parasites or beneficial mutualists? What is the evolutionary origin of hitchers? Are there key advantages associated with hitchers' lifestyle that justify their unexpected abundance? And why are hitchers systematically smaller than their helpers? In this essay, we start answering these questions and point ways ahead for understanding the principles, origin, mechanisms, and impact of hitchers in bacterial ecology and evolution.

ARGV: 3D genome structure exploration using augmented reality

Over the past two decades, scientists have increasingly realized the importance of the three-dimensional (3D) genome organization in regulating cellular activity. Hi-C and related experiments yield 2D contact matrices that can be used to infer 3D models of chromosome structure. Visualizing and analyzing genomes in 3D space remains challenging. Here, we present ARGV, an augmented reality 3D Genome Viewer. ARGV contains more than 350 pre-computed and annotated genome structures inferred from Hi-C and imaging data. It offers interactive and collaborative visualization of genomes in 3D space, using standard mobile phones or tablets. A user study comparing ARGV to existing tools demonstrates its benefits.

3D genomic features across >50 diverse cell types reveal insights into the genomic architecture of childhood obesity.

The prevalence of childhood obesity is increasing worldwide, along with the associated common comorbidities of type 2 diabetes and cardiovascular disease in later life. Motivated by evidence for a strong genetic component, our prior genome-wide association study (GWAS) efforts for childhood obesity revealed 19 independent signals for the trait; however, the mechanism of action of these loci remains to be elucidated. To molecularly characterize these childhood obesity loci we sought to determine the underlying causal variants and the corresponding effector genes within diverse cellular contexts. Integrating childhood obesity GWAS summary statistics with our existing 3D genomic datasets for 57 human cell types, consisting of high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq, and RNA-seq, we applied stratified LD score regression and calculated the proportion of genome-wide SNP heritability attributable to cell type-specific features, revealing pancreatic alpha cell enrichment as the most statistically significant. Subsequent chromatin contact-based fine-mapping was carried out for genome-wide significant childhood obesity loci and their linkage disequilibrium proxies to implicate effector genes, yielded the most abundant number of candidate variants and target genes at the BDNF, ADCY3, TMEM18 and FTO loci in skeletal muscle myotubes and the pancreatic beta-cell line, EndoC-BH1. One novel implicated effector gene, ALKAL2 - an inflammation-responsive gene in nerve nociceptors - was observed at the key TMEM18 locus across multiple immune cell types. Interestingly, this observation was also supported through colocalization analysis using expression quantitative trait loci (eQTL) derived from the Genotype-Tissue Expression (GTEx) dataset, supporting an inflammatory and neurologic component to the pathogenesis of childhood obesity. Our comprehensive appraisal of 3D genomic datasets generated in a myriad of different cell types provides genomic insights into pediatric obesity pathogenesis.

A Comparison of Structural Variant Calling from Short-Read and Nanopore-Based Whole-Genome Sequencing Using Optical Genome Mapping as a Benchmark.

The identification of structural variants (SVs) in genomic data represents an ongoing challenge because of difficulties in reliable SV calling leading to reduced sensitivity and specificity. We prepared high-quality DNA from 9 parent-child trios, who had previously undergone short-read whole-genome sequencing (Illumina platform) as part of the Genomics England 100,000 Genomes Project. We reanalysed the genomes using both Bionano optical genome mapping (OGM; 8 probands and one trio) and Nanopore long-read sequencing (Oxford Nanopore Technologies [ONT] platform; all samples). To establish a "truth" dataset, we asked whether rare proband SV calls (n = 234) made by the Bionano Access (version 1.6.1)/Solve software (version 3.6.1_11162020) could be verified by individual visualisation using the Integrative Genomics Viewer with either or both of the Illumina and ONT raw sequence. Of these, 222 calls were verified, indicating that Bionano OGM calls have high precision (positive predictive value 95%). We then asked what proportion of the 222 true Bionano SVs had been identified by SV callers in the other two datasets. In the Illumina dataset, sensitivity varied according to variant type, being high for deletions (115/134; 86%) but poor for insertions (13/58; 22%). In the ONT dataset, sensitivity was generally poor using the original Sniffles variant caller (48% overall) but improved substantially with use of Sniffles2 (36/40; 90% and 17/23; 74% for deletions and insertions, respectively). In summary, we show that the precision of OGM is very high. In addition, when applying the Sniffles2 caller, the sensitivity of SV calling using ONT long-read sequence data outperforms Illumina sequencing for most SV types.

Breast Cancer High-Penetrance Genes BRCA1 and BRCA2 Mutations Using Next-Generation Sequencing Among Iraqi Kurdish Women.

Background BRCA1andBRCA2genesare the main high-penetrance genes that are responsible for most cases of inherited breast cancer. The present study aimed to detect the frequencies of inherited breast cancer caused by BRCA1 and BRCA2 genes among Kurdish breast cancer patients, including all the exome of these two genes, using next-generation sequencing (NGS). Methodology Seventy women who were diagnosed with breast cancer and registered at Nanakali Hospital in Erbil, Iraq, were included. Blood samples were collected for molecular testing (polymerase chain reaction (PCR)) targeting all exomes of BRCA1 and BRCA2 genes. All exome regions are sequenced by NGS using the Miseq system (Illumina Inc., San Diego, CA). Obtained data were visualized using Integrative Genomics Viewer (IGV 2.3 Software, Broad Institute, Cambridge, MA). Data were interpreted based on the National Center for Biotechnology Information (NCBI), Clinically Relevant Variation (ClinVar) archives, and other databases. Results Among 70 samples, more than forty-two variants have been detected, 20 on BRCA1 and 22 on BRCA2. Regarding clinical significance, six (14.28%) variants were pathogenic, four of them on theBRCA1 gene, which were: c.3607C>T, c.3544C>T, c.68_69del, and c.224_227delAAAG, and two pathogenic variants were on BRCA2 gene: c.100G>T, and c.1813delA. Also, two (4.76%) variants were conflict interpretations of pathogenicity, one (2.38%) was a variant of uncertain significant VUS, and the rest 29 (69%) variants were benign.In addition, four new variants (three in BRCA1 and one in BRCA2 gene), never previously reported, were identified. Conclusions In conclusion, analyzing the BRCA1/2 genes provide a better prediction for the risk of developing breast cancer in the future. Variant types and frequencies differ among different populations and ethnicities, the common mutations worldwide may not be prevalent in the Kurdish population. The current research findings will be useful for future screening studies of these two genes in the Kurdish population.

The NCBI Comparative Genome Viewer (CGV) is an interactive visualization tool for the analysis of whole-genome eukaryotic alignments.

We report a new visualization tool for analysis of whole-genome assembly-assembly alignments, the Comparative Genome Viewer (CGV) (https://ncbi.nlm.nih.gov/genome/cgv/). CGV visualizes pairwise same-species and cross-species alignments provided by National Center for Biotechnology Information (NCBI) using assembly alignment algorithms developed by us and others. Researchers can examine large structural differences spanning chromosomes, such as inversions or translocations. Users can also navigate to regions of interest, where they can detect and analyze smaller-scale deletions and rearrangements within specific chromosome or gene regions. RefSeq or user-provided gene annotation is displayed where available. CGV currently provides approximately 800 alignments from over 350 animal, plant, and fungal species. CGV and related NCBI viewers are undergoing active development to further meet needs of the research community in comparative genome visualization.

Genetic diversity and phylogeographic dynamics of avihepadnavirus: a comprehensive full-length genomic view.

Avihepadnavirus is a genus of the Hepadnaviridae family. It primarily infects birds, including species of duck, geese, cranes, storks, and herons etc. To understand the genetic relatedness and evolutionary diversity among avihepadnavirus strains, a comprehensive analysis of the available 136 full-length viral genomes (n = 136) was conducted. The genomes were classified into two major genotypes, i.e., GI and GII. GI viruses were further classified into 8 sub-genotypes including DHBV-I (duck hepatitis B virus-I), DHBV-II (Snow goose Hepatitis B, SGHBV), DHBV-III, RGHBV (rossgoose hepatitis B virus), CHBV (crane hepatitis B virus), THBV (Tinamou hepatitis B virus), STHBV (stork hepatitis B virus), and HHBV (Heron hepatitis B virus). DHBV-I contains two sub-clades DHBV-Ia and DHBV-Ib. Parrot hepatitis B virus (PHBV) stains fall into GII which appeared as a separate phylogenetic branch/clade. All the subtypes of viruses in GI and GII seem to be genetically connected with viruses of DHBV-I by multiple mutational steps in phylogeographic analysis. Furthermore, 16 potential recombination events among different sub-genotypes in GI and one in GII were identified, but none of which is inter-genotypic between GI and GII. Overall, the results provide a whole picture of the genetic relatedness of avihepadnavirus strains, which may assist in the surveillance of virus spreading.

SNP-SVant: A Computational Workflow to Predict and Annotate Genomic Variants in Organisms Lacking Benchmarked Variants.

Whole-genome sequencing is widely used to investigate population genomic variation in organisms of interest. Assorted tools have been independently developed to call variants from short-read sequencing data aligned to a reference genome, including single nucleotide polymorphisms (SNPs) and structural variations (SVs). We developed SNP-SVant, an integrated, flexible, and computationally efficient bioinformatic workflow that predicts high-confidence SNPs and SVs in organisms without benchmarked variants, which are traditionally used for distinguishing sequencing errors from real variants. In the absence of these benchmarked datasets, we leverage multiple rounds of statistical recalibration to increase the precision of variant prediction. The SNP-SVant workflow is flexible, with user options to tradeoff accuracy for sensitivity. The workflow predicts SNPs and small insertions and deletions using the Genome Analysis ToolKit (GATK) and predicts SVs using the Genome Rearrangement IDentification Software Suite (GRIDSS), and it culminates in variant annotation using custom scripts. A key utility of SNP-SVant is its scalability. Variant calling is a computationally expensive procedure, and thus, SNP-SVant uses a workflow management system with intermediary checkpoint steps to ensure efficient use of resources by minimizing redundant computations and omitting steps where dependent files are available. SNP-SVant also provides metrics to assess the quality of called variants and converts between VCF and aligned FASTA format outputs to ensure compatibility with downstream tools to calculate selection statistics, which are commonplace in population genomics studies. By accounting for both small and large structural variants, users of this workflow can obtain a wide-ranging view of genomic alterations in an organism of interest. Overall, this workflow advances our capabilities in assessing the functional consequences of different types of genomic alterations, ultimately improving our ability to associate genotypes with phenotypes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Predicting single nucleotide polymorphisms and structural variations Support Protocol 1: Downloading publicly available sequencing data Support Protocol 2: Visualizing variant loci using Integrated Genome Viewer Support Protocol 3: Converting between VCF and aligned FASTA formats.

An evolutionary view of the Fusarium core genome

Fusarium, a member of the Ascomycota fungi, encompasses several pathogenic species significant to plants and animals. Some phytopathogenic species have received special attention due to their negative economic impact on the agricultural industry around the world. Traditionally, identification and taxonomic analysis of Fusarium have relied on morphological and phenotypic features, including the fungal host, leading to taxonomic conflicts that have been solved using molecular systematic technologies. In this work, we applied a phylogenomic approach that allowed us to resolve the evolutionary history of the species complexes of the genus and present evidence that supports the F. ventricosum species complex as the most basal lineage of the genus. Additionally, we present evidence that proposes modifications to the previous hypothesis of the evolutionary history of the F. staphyleae, F. newnesense, F. nisikadoi, F. oxysporum, and F. fujikuroi species complexes. Evolutionary analysis showed that the genome GC content tends to be lower in more modern lineages, in both, the whole-genome and core-genome coding DNA sequences. In contrast, genome size gain and losses are present during the evolution of the genus. Interestingly, core genome duplication events positively correlate with genome size. Evolutionary and genome conservation analysis supports the F3 hypothesis of Fusarium as a more compact and conserved group in terms of genome conservation. By contrast, outside of the F3 hypothesis, the most basal clades only share 8.8% of its genomic sequences with the F3 clade.

Abstract 4352: A high rate of episomal HPV16 is present in head and neck squamous cell carcinoma tumors by long-read whole genome sequencing

Abstract Persistent Human Papillomavirus (HPV) infection causes an increased risk of Head and Neck Squamous Cell Carcinoma (HNSCC). However, a comprehensive understanding of the intricate molecular events within virus-positive tumors remains a critical knowledge gap. Therefore, we used long-read sequencing to study the intricacies of HPV integration events within the genomic landscape of HPV+ HNSCC tumors. A total of 16 tissue biopsies, including 10 tumors and 6 matched adjacent normal tissues from histopathologically confirmed HNSCC subjects were sequenced by LSK114 chemistry of Oxford Nanopore Technology with an average coverage of 30X and 10X, respectively. The predicted mitochondrial haplotype from Mitomaster confirmed that the tumor/normal pair was from the same individual. The HPV integration status was determined by aligning fastq_pass files at the Cancer Genomics Cloud and viewing the breakpoints in the Integrated Genome Viewer. HPV lineage and sub-lineage classification was determined using ClustalOmega. We detected HPV16 in 8/10 tumors, with sublineages A1 and A2 accounting for 38% (3/8) and D3 for 25% (2/8). One tumor sample had a very low HPV load with only one HPV16 sequence read and low detection by PCR. Interestingly, we found that 5/8 HPV positive tumors had episomal HPV16 with different states. So, two tumors had intact HPV episomes, two more had either a small deletion or an insertion in the episomal HPV, and the last one had a rearranged episomal multimer (Table 1). The remaining three tumors had integrated HPV16: one with type 1 integrations at 11q12.13 and two others with type 2 integrations at chr1q42.12 and chr14q21.1. Our results indicate that episomal HPV16 is more prevalent than integrated HPV16 in primary HPV+ HNSCC samples. These findings provide new insights into the molecular mechanisms and consequences of HPV infection in HNSCC. Table 1: HPV characterization in HNSCC studied tissue biopsies. Patient ID Race HPV status HPV sublineages Predicted Haplotype HPV pattern #PID_1 Caucasian HPV16 + A2 T2b (T2b) Type 2 Integration (chr1q42.12) #PID_2 - HPV- - I1a (I1a1b) - #PID_3 Caucasian HPV16 + A1 H1q (H1q) Episomal (rearranged multimer) PID_4 African American HPV16 + A1 - Episomal (deletion of 636bp from 4892 to 5528) #PID_5 - HPV16 + A2 H1 (H1+16189) Type 1 Integration (complex chr11q12.13) *#PID_6 Caucasian HPV16 + - T2b (T2b) - PID_7 Caucasian HPV16 + D3 - Episomal #PID_8 African American HPV16 + D3 L1c (L1c3a) Type 2 Integration (chr14q21.1) PID_9 African American HPV16 + A2 - Episomal (insertion of 257bp from 7310-7567) PID_10 Caucasian HPV16 + A1 - Episomal *Very weakly HPV16 positive; #Tumor-normal pair Citation Format: Sonam Tulsyan, Vera Mukhina, Isabel Rodriguez, Subhendu R. Choudhury, Erin Allor, Kyle Hatte, Michael Dean, Daria A. Gaykalova. A high rate of episomal HPV16 is present in head and neck squamous cell carcinoma tumors by long-read whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4352.

Abstract 7426: The GenePattern ecosystem for cancer bioinformatics

Abstract As the availability of genomic data and analysis tools from large-scale cancer initiatives continues to increase, with single-cell studies adding new dimensions to the potential scientific insights, the need has become more urgent for a software environment that supports the rapid pace of cancer data science. The GenePattern ecosystem, first introduced in 2004 and updated continually to support the changing needs of cancer genomics research, supports the analytical, computational, and reproducibility needs of the world-wide cancer analysis community.The GenePattern server, available at www.genepattern.org, provides hundreds of analysis methods for scientists at all levels of computational sophistication, with the only requirement for use being a web browser. The GenePattern server offers bulk and single-cell RNA-seq, copy number variation, flow cytometry, network analysis, general machine learning, gene set enrichment analysis, proteomics, and many other modalities. Analysis steps can be linked together into pipelines that can then be edited, shared, and made public. All parameters of an analysis, including the code version, are recorded so that an analytical result can be reproduced at any point in the future.The GenePattern Notebook system, notebook.genepattern.org, is an integration of the Jupyter Notebook environment with the GenePattern server. It combines the research narrative capabilities of Jupyter with the non-programming approach and breadth of analyses available in GenePattern. Scientists using GenePattern Notebook can create documents that include richly-formatted text and multimedia, executable code, and GenePattern analyses. A single GenePattern notebook can therefore comprise a sophisticated analytical workflow that runs on multiple remote servers.We have recently expanded GenePattern Notebook into a new notebook environment, Genomics to Notebook, g2nb.org, which adds the ability to include analyses on any public Galaxy server as well as using the Integrative Genomics Viewer (IGV) and Cytoscape within notebooks. Each analysis or visualization appears a cell within a notebook, preserving the accessibility and ease of use of the notebook metaphor while retaining the essential aspects of each tool’s user interface. When run, the entire analysis appears to execute seamlessly within the notebook. The online workspace also includes a library of featured genomic analysis notebooks, including templates for common analysis tasks as well as cancer-specific research scenarios and compute-intensive methods. Scientists can easily copy these notebooks, use them as is, or adapt them for their research purposes. Citation Format: Michael M. Reich, Thorin Tabor, John Liefeld, Edwin Huang, Forrest Kim, Jill P. Mesirov. The GenePattern ecosystem for cancer bioinformatics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7426.

Performance and characterization of 94 identity-informative SNPs in Northern Han Chinese using ForenSeq ™ DNA signature prep kit

Target and flanking region (FR) variation at 94 identity-informative SNPs (iSNPs) are investigated in 635 Northern Han Chinese using the ForenSeq DNA Signature Prep Kit on the MiSeq FGx Forensic Genomics System. The dataset presents the following performance characteristics (average values): ≥60% bases with a quality score of 20 or higher (%≥ Q20); >700 × of depth of coverage (DoC) from both Sample Details Reports and Flanking Region Reports; >80% of effective reads; ≥60% of allele coverage ratio (ACR); and ≥70% of inter-locus balance, while some stable low-performance characteristics are also observed: low DoC at rs1736442, rs1031825, rs7041158, rs338882, rs2920816, rs1493232, rs719366, and rs2342747; high noise at rs891700; and imbalanced ACR at rs6955448 and rs338882. The average amplicon length is 69 bp, suitable for detecting degraded samples. Bioinformatic concordance achieves 99.99% between the ForenSeq Universal Analysis Software (UAS) and the Integrative Genomic Viewer (IGV) inspection. Discordance results from flanking region deletions of rs10776839, rs8078417, rs2831700, and rs1454361. Due to FR variants within amplicons detected by massively parallel sequencing (MPS), the increases in the number of unique alleles, effective alleles (Ae), and observed heterozygosity (Hobs) are 46.81%, 4.51%, and 3.29%, respectively. Twelve FR variants are first reported to dbSNP, such as rs1252699848, rs1665500714, rs1771121532, rs2097285015, rs1851671415, rs2045669877, rs2046758811, rs2044248635, rs1251308240, rs1968822112, rs1981638299, and rs1341756746. All 94 iSNPs from target and amplicon data are in Hardy-Weinberg equilibrium (HWE) and independent within autosomes. As expected, forensic parameters from the amplicon data increase significantly on the combined power of discrimination (CPD = 1 – 3.9876 × 10−38) and the combined power of exclusion (CPE = 1 – 6.6690 × 10−8). Additionally, the power of the system effectiveness (CPD = 1 − 6.7054 × 10−72 and CPE = 1 − 4.4719 × 10−20) with sequence-based 27 autosomal STRs and 94 iSNP amplicons in combination is substantially improved compared to one type of marker alone. In conclusion, we have established a traditional length-based and current sequence-based reference database with 58 STRs and 94 iSNPs in the Northern Han Chinese population. We hope these data can serve as a solid reference and foundation for forensic practice.

Analyzing Colorectal Cancer at the Molecular Level through Next-generation Sequencing in Erbil City

Colorectal cancer (CRC) ranks as the third leading cause of cancer-related deaths globally. It is characterized as a genomic disorder marked by diverse genomic anomalies, including point mutations, genomic rearrangements, gene fusions, and alterations in chromosomal copy numbers. This research aims to identify previously undisclosed genetic variants associated with an increased risk of CRC by employing next-generation sequencing technology. Genomic DNA was extracted from blood specimens of five CRC patients. The sequencing data of the samples are utilized for variant identification. In addition, the Integrative Genomic Viewer software (IGV) is used to visualize the identified variants. Furthermore, various in silico tools, including Mutation Taster and Align GVGD, are used to predict the potential impact of mutations on structural features and protein function. Based on the findings of this research, 12 different genetic variations are detected among individuals with CRC. Inherited variations are located within the following genes: MSH6, MSH2, PTPRJ, PMS2, TP53, BRAF, APC, and PIK3CA.

Cold Tumour Phenotype Explained Through Whole Genome Sequencing in Clinical Nasopharyngeal Cancer: A Preliminary Study.

Nasopharyngeal cancer (NPC) is a complex cancer due to its unique genomic features and association with the Epstein-Barr virus (EBV). Despite therapeutic advancements, NPC prognosis remains poor, necessitating a deeper understanding of its genomics. Here, we present a comprehensive whole genome sequencing (WGS) view of NPC genomics and its correlation with the phenotype. This study involved WGS of a clinical NPC biopsy specimen. Sequencing was carried out using a long read sequencer from Oxford Nanopore. Analysis of the variants involved correlation with the phenotype of NPC. A loss of genes within chromosome 6 from copy number variation (CNV) was found. The lost genes included HLA-A, HLA-B, and HLA-C, which work in the antigen presentation process. This loss of the major histocompatibility complex (MHC) apparatus resulted in the tumour's ability to evade immune recognition. The tumour exhibited an immunologically "cold" phenotype, with mild tumour-infiltrating lymphocytes, supporting the possible etiology of loss of antigen presentation capability. Furthermore, the driver mutation PIK3CA gene was identified along with various other gene variants affecting numerous signaling pathways. Comprehensive WGS was able to detect various mutations and genomic losses, which could explain tumour progression and immune evasion ability. Furthermore, the study identified the loss of other genes related to cancer and immune pathways, emphasizing the complexity of NPC genomics. In conclusion, this study underscores the significance of MHC class I gene loss and its probable correlation with the cold tumour phenotype observed in NPC.

Bilateral Peters' anomaly, aniridia and Wilms tumour (WAGR syndrome) in monozygotic twins.

This study reports the bilateral association of Peters' anomaly and congenital aniridia in monozygotic twins subsequently diagnosed with Wilms tumour (WAGR syndrome). Two monozygotic female twins were referred at age 2 months with bilateral corneal opacity. A diagnosis of Peters' anomaly associated to aniridia was made in both eyes of both twins. Physical examination and ultrasonography were carried out at 12 months of age to explore the possibility of WAGR-related anomalies, specifically Wilms tumour. DNA were isolated and subjected to whole exome sequencing. Peters' anomaly associated to aniridia in both eyes as well as bilateral Wilms tumour in both children were diagnosed. Exome analyses showed a large heterozygous deletion encompassing 6 648 473 bp in chromosome 11p13, using Integrative Genomics Viewer and AnnotSV software. WAGR syndrome is a rare contiguous gene deletion syndrome with a greater risk of developing Wilms tumour associated with Peters' anomaly and congenital aniridia. However, co-occurrence of both anomalies was rarely reported in twins, and never in both eyes of monozygotic twins. Here, we report the bilateral association of Peters' anomaly and congenital aniridia in monozygotic twins with WAGR syndrome.

Control-FREEC viewer: a tool for the visualization and exploration of copy number variation data

BackgroundCopy number alterations (CNAs) are genetic changes commonly found in cancer that involve different regions of the genome and impact cancer progression by affecting gene expression and genomic stability. Computational techniques can analyze copy number data obtained from high-throughput sequencing platforms, and various tools visualize and analyze CNAs in cancer genomes, providing insights into genetic mechanisms driving cancer development and progression. However, tools for visualizing copy number data in cancer research have some limitations. In fact, they can be complex to use and require expertise in bioinformatics or computational biology. While copy number data analysis and visualization provide insights into cancer biology, interpreting results can be challenging, and there may be multiple explanations for observed patterns of copy number alterations.ResultsWe created Control-FREEC Viewer, a tool that facilitates effective visualization and exploration of copy number data. With Control-FREEC Viewer, experimental data can be easily loaded by the user. After choosing the reference genome, copy number data are displayed in whole genome or single chromosome view. Gain or loss on a specific gene can be found and visualized on each chromosome. Analysis parameters for subsequent sessions can be stored and images can be exported in raster and vector formats.ConclusionsControl-FREEC Viewer enables users to import and visualize data analyzed by the Control-FREEC tool, as well as by other tools sharing a similar tabular output, providing a comprehensive and intuitive graphical user interface for data visualization.

Global exact optimisations for chloroplast structural haplotype scaffolding

BackgroundScaffolding is an intermediate stage of fragment assembly. It consists in orienting and ordering the contigs obtained by the assembly of the sequencing reads. In the general case, the problem has been largely studied with the use of distances data between the contigs. Here we focus on a dedicated scaffolding for the chloroplast genomes. As these genomes are small, circular and with few specific repeats, numerous approaches have been proposed to assemble them. However, their specificities have not been sufficiently exploited.ResultsWe give a new formulation for the scaffolding in the case of chloroplast genomes as a discrete optimisation problem, that we prove the decision version to be NP\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\mathcal{NP}$$\\end{document}-Complete. We take advantage of the knowledge of chloroplast genomes and succeed in expressing the relationships between a few specific genomic repeats in mathematical constraints. Our approach is independent of the distances and adopts a genomic regions view, with the priority on scaffolding the repeats first. In this way, we encode the structural haplotype issue in order to retrieve several genome forms that coexist in the same chloroplast cell. To solve exactly the optimisation problem, we develop an integer linear program that we implement in Python3 package khloraascaf. We test it on synthetic data to investigate its performance behaviour and its robustness against several chosen difficulties.ConclusionsWe succeed to model biological knowledge on genomic structures to scaffold chloroplast genomes. Our results suggest that modelling genomic regions is sufficient for scaffolding repeats and is suitable for finding several solutions corresponding to several genome forms.