Whole Genome Sequencing Samples Research Articles

Retinoic Acid-Induced 1 gene variants associated with Smith–Magenis syndrome circadian phenotypes enriched in autism spectrum disorder: whole-genome sequencing study

BackgroundThis study aimed to characterize the frequency of RAI1 genetic aberrations associated with Smith–Magenis syndrome (SMS), in a large cohort of autism spectrum disorder (ASD) whole-genome sequencing samples. We aimed to determine the frequencies of RAI1 single-nucleotide variants (SNVs) and copy number variants (CNVs).ResultsWe report a 2.5 × enrichment of the major deletion and a > 5 × enrichment of the frameshift variants as compared to the known prevalence of SMS 1/15,000. Additionally, we report a significant enrichment of RAI1 rare missense variants in ASD subjects with respect to controls (54 variants/6080 ASD subjects and 6 variants/2541 controls, p-value < 0.002, OR 3.78, CI 1.62–8–81).ConclusionsThe SMS phenotype including circadian dysregulation and associated sleep disturbances is mainly caused by RAI1 haploinsufficiency. Sleep disturbances as seen in SMS may overlap in ASD, especially in patients with consequential variants in RAI1 gene.

A genome-wide spectrum of tandem repeat expansions in 338,963 humans

The Genome Aggregation Database (gnomAD), widely recognized as the gold-standard reference map of human genetic variation, has largely overlooked tandem repeat (TR) expansions, despite the fact that TRs constitute ∼6% of our genome and are linked to over 50 human diseases. Here, we introduce the TR-gnomAD (https://wlcb.oit.uci.edu/TRgnomAD), a biobank-scale reference of 0.86 million TRs derived from 338,963 whole-genome sequencing (WGS) samples of diverse ancestries (39.5% non-European samples). TR-gnomAD offers critical insights into ancestry-specific disease prevalence using disparities in TR unit number frequencies among ancestries. Moreover, TR-gnomAD is able to differentiate between common, presumably benign TR expansions, which are prevalent in TR-gnomAD, from those potentially pathogenic TR expansions, which are found more frequently in disease groups than within TR-gnomAD. Together, TR-gnomAD is an invaluable resource for researchers and physicians to interpret TR expansions in individuals with genetic diseases.

Abstract 7014: Integrative DNA methylation cancer profiling reveals patterns associated with tumor progression and treatment response

Abstract DNA methylation plays a critical role in tumor progression and response to chemotherapy. Frequently dysregulated during tumor development, it is closely intertwined with mixed effects from patient epigenetic background, tissue context, and treatment pressure. Here, we leveraged methylomic and transcriptomic profiling of 400 whole-genome sequencing samples from 125 patients with ovarian high-grade serous carcinoma (HGSC), belonging to the prospective, longitudinal, multi-region DECIDER trial (NCT04846933). This cohort included 120 samples from tubo-ovarian tumors, 209 samples from intra-abdominal metastases, and 71 ascites, representing all major sites of HGSC metastases. We profiled cancer methylome across tissues, patient groups, and various anticancer treatments using decomposition method, which allows separating the effects of tumor microenvironment and treatment, tissue, and patient specific changes, thus, facilitating integration of the heterogeneous collection of samples. First, we systematically examined the tissue-specific carcinogenesis and characterized methylomes of all types of metastases and excess fluid accumulations in the abdominal cavity. In our analyses, the cancer spread from tubo-ovarian sites to metastatic deposits involved gradual loss of DNA methylation and increase in transcriptomic levels, possibly indicating stemness. Specifically, several members of the ATP-binding-cassette (ABC) family of transporters, as well as receptor tyrosine kinases were frequently hypomethylated in bowel and omentum. The methylome of ascites showed most commonality to the tubo-ovarian tumors and peritoneum. Our results confirmed that several well-known tumor-associated genes, such as PTEN, GSK3B, ESR1, FGFR2, TNC, BCL6, HIF1A, NOTCH2 are regulated epigenetically, and this dysregulation stems early from the sites of origin. Next, we built methylomic profiles of patients with various treatment responses. Direct comparisons between good and poor responders revealed pronounced methylation differences, with refractory disease being associated with severely aberrated methylome prior to chemotherapy treatment. Neoadujuvant chemotherapy resulted in very few changes to these epigenetic aberrations and no acquired mechanisms, suggesting that intrinsic methylomes of poor responders accommodate multiple drivers for EMT, hypoxia, and chronic inflammation to resist chemotherapy-induced cell death already prior to treatment. In conclusion, using a large whole-genome sequencing DNA methylation dataset we elucidated the major underlying epigenetic mechanisms of tumorigenesis and platinum resistance, which provide possible drug targets for improving platinum sensitivity. Our multi-omics analyses enabled the identification of aberrated patterns that drive transcriptional changes, shedding light on epigenetic landscape of HGSC. Citation Format: Alexandra Lahtinen, Giovanni Marchi, Daria Afenteva, Kari Lavikka, Susanna Holmström, Elsi Pöllänen, Giulia Micoli, Ilari Maarala, Yilin Li, Taru A. Muranen, Jaana Oikkonen, Sakari Hietanen, Anni Virtanen, Johanna Hynninen, Antti Häkkinen, Sampsa Hautaniemi. Integrative DNA methylation cancer profiling reveals patterns associated with tumor progression and treatment response [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 7014.

Genomic insights into local-scale evolution of ocular Chlamydia trachomatis strains within and between individuals in Gambian trachoma-endemic villages.

Trachoma, a neglected tropical disease caused by Chlamydia trachomatis (Ct) serovars A-C, is the leading infectious cause of blindness worldwide. Africa bears the highest burden, accounting for over 86 % of global trachoma cases. We investigated Ct serovar A (SvA) and B (SvB) whole genome sequences prior to the induction of mass antibiotic drug administration in The Gambia. Here, we explore the factors contributing to Ct strain diversification and the implications for Ct evolution within the context of ocular infection. A cohort study in 2002-2003 collected ocular swabs across nine Gambian villages during a 6 month follow-up study. To explore the genetic diversity of Ct within and between individuals, we conducted whole-genome sequencing (WGS) on a limited number (n=43) of Ct-positive samples with an omcB load ≥10 from four villages. WGS was performed using target enrichment with SureSelect and Illumina paired-end sequencing. Out of 43 WGS samples, 41 provided sufficient quality for further analysis. ompA analysis revealed that 11 samples had highest identity to ompA from strain A/HAR13 (NC_007429) and 30 had highest identity to ompA from strain B/Jali20 (NC_012686). While SvB genome sequences formed two distinct village-driven subclades, the heterogeneity of SvA sequences led to the formation of many individual branches within the Gambian SvA subclade. Comparing the Gambian SvA and SvB sequences with their reference strains, Ct A/HAR13 and Ct B/Jali20, indicated an single nucleotide polymorphism accumulation rate of 2.4×10-5 per site per year for the Gambian SvA and 1.3×10-5 per site per year for SvB variants (P<0.0001). Variant calling resulted in a total of 1371 single nucleotide variants (SNVs) with a frequency >25 % in SvA sequences, and 438 SNVs in SvB sequences. Of note, in SvA variants, highest evolutionary pressure was recorded on genes responsible for host cell modulation and intracellular survival mechanisms, whereas in SvB variants this pressure was mainly on genes essential for DNA replication/repair mechanisms and protein synthesis. A comparison of the sequences between observed separate infection events (4-20 weeks between infections) suggested that the majority of the variations accumulated in genes responsible for host-pathogen interaction such as CTA_0166 (phospholipase D-like protein), CTA_0498 (TarP) and CTA_0948 (deubiquitinase). This comparison of Ct SvA and SvB variants within a trachoma endemic population focused on their local evolutionary adaptation. We found a different variation accumulation pattern in the Gambian SvA chromosomal genes compared with SvB, hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies.

A compendium of genetic regulatory effects across pig tissues

The Farm Animal Genotype-Tissue Expression (FarmGTEx) project has been established to develop a public resource of genetic regulatory variants in livestock, which is essential for linking genetic polymorphisms to variation in phenotypes, helping fundamental biological discovery and exploitation in animal breeding and human biomedicine. Here we show results from the pilot phase of PigGTEx by processing 5,457 RNA-sequencing and 1,602 whole-genome sequencing samples passing quality control from pigs. We build a pig genotype imputation panel and associate millions of genetic variants with five types of transcriptomic phenotypes in 34 tissues. We evaluate tissue specificity of regulatory effects and elucidate molecular mechanisms of their action using multi-omics data. Leveraging this resource, we decipher regulatory mechanisms underlying 207 pig complex phenotypes and demonstrate the similarity of pigs to humans in gene expression and the genetic regulation behind complex phenotypes, supporting the importance of pigs as a human biomedical model.

Preselecting Variants from Large-Scale Genome-Wide Association Study Meta-Analyses Increases the Genomic Prediction Accuracy of Growth and Carcass Traits in Large White Pigs.

Preselected variants associated with the trait of interest from genome-wide association studies (GWASs) are available to improve genomic prediction in pigs. The objectives of this study were to use preselected variants from a large GWAS meta-analysis to assess the impact of single-nucleotide polymorphism (SNP) preselection strategies on genome prediction of growth and carcass traits in pigs. We genotyped 1018 Large White pigs using medium (50k) SNP arrays and then imputed SNPs to sequence level by utilizing a reference panel of 1602 whole-genome sequencing samples. We tested the effects of different proportions of selected top SNPs across different SNP preselection strategies on genomic prediction. Finally, we compared the prediction accuracies by employing genomic best linear unbiased prediction (GBLUP), genomic feature BLUP and three weighted GBLUP models. SNP preselection strategies showed an average improvement in accuracy ranging from 0.3 to 2% in comparison to the SNP chip data. The accuracy of genomic prediction exhibited a pattern of initial increase followed by decrease, or continuous decrease across various SNP preselection strategies, as the proportion of selected top SNPs increased. The highest level of prediction accuracy was observed when utilizing 1 or 5% of top SNPs. Compared with the GBLUP model, the utilization of estimated marker effects from a GWAS meta-analysis as SNP weights in the BLUP|GA model improved the accuracy of genomic prediction in different SNP preselection strategies. The new SNP preselection strategies gained from this study bring opportunities for genomic prediction in limited-size populations in pigs.

ADSP Whole Genome Sequencing (WGS) Release 4 Data Update from Genome Center for Alzheimer’s Disease

AbstractBackgroundThe Genome Center for Alzheimer’s Disease (GCAD) coordinates the integration of all available Alzheimer’s disease (AD) relevant whole genome sequencing (WGS) data with the goal of identifying AD risk or protective genetic variants and eventual therapeutic targets. The WGS datasets are generated through collaboration between investigators from the Alzheimer’s Disease Sequencing Project (ADSP) and GCAD. With the goal of minimizing data heterogeneity, introduced by different sequencing protocols and assays, GCAD processes all samples using standardized pipelines and performs quality control (QC)/quality assurance (QA) checks.MethodsRaw sequencing data (FASTQs or BAMs) were aligned to GRCh38/hg38 by BWA, and variant calling and joint genotyping on single nucleotide variants (SNVs), insertions and deletions (indels), were done by GATK. Structural variants (SVs) were called per sample using the Smoove, Manta, and Strelka packages. Preliminary QA checks including sex check, contamination, and genotype concordance were performed followed by QC per ADSP protocol to evaluate the quality of samples and variants. To facilitate access and usage of massive joint‐genotype called VCF files, a compact version for storing variant info and sample genotypes only was released first.ResultsWe dropped 275 (0.7%) samples of poor coverage (&lt;20×), and we flagged 219 (0.6%) samples that were of borderline quality. As a result, the dataset (ADSP Release 4, 2022) includes 36,361 genomes from 40 diverse cohorts with 4 major ancestries: 16,573 Non‐Hispanic Whites, 11,358 Hispanics; 5,422 African Americans; and 2,802 Asians. Data are deeply sequenced (average genome coverage: 40x). All samples’ CRAMs and gVCFs from GATK were deposited into NIAGADS Data Sharing Service (DSS) (https://dss.niagads.org/) for public distribution. Joint‐genotyped called VCFs are undergoing a full QC/annotation process and will be made available. This joint‐genotyped called VCF contains &gt;362M bi‐allelic variants, &gt;58M multi‐allelic variants, with 95% of variants remaining after QC. SV calling is ongoing and data will be ready prior to the conference.ConclusionThe ADSP and GCAD generate high quality SNVs, indels and SV calls. Currently GCAD is preparing the next release of ∼60,000 more ancestrally‐diverse WGS samples sequenced primarily through the ADSP Follow‐Up Study, which we anticipate will be released in 2023 to greatly benefit the AD genetics community.

Routine Whole Genome Sequencing Offers a More Comprehensive Assessment of IG/TCR Gene Rearrangements for MRD Targets in Acute Lymphoblastic Leukemia - Data from the First 62 Cases from the NHS England Genomic Medicine Service

Introduction Minimal residual disease (MRD) monitoring is fundamental for risk stratification and treatment in acute lymphoblastic leukemia (ALL). Sensitive molecular assay requires presentation samples to be screened for immunoglobulin and T-cell receptor (IG/TCR) gene rearrangements. Previously samples were screened by PCR, heteroduplex analysis and Sanger sequencing to identify clone defining IG/TCR gene rearrangements. Subsequently these sequences were used to develop qPCR assays for MRD follow-up monitoring. More recently, targeted IG/TCR high-throughput sequencing (HTS) has been used with similar panels to identify gene rearrangements. Both these methods have limitations, in that only rearrangements covered by the panels are detected, and the techniques are labour intensive and expensive. Whole genome sequencing (WGS) has potential to discover all IG/TCR rearrangements in a single assay giving greater potential to detect markers for follow up MRD monitoring. In England all patients with ALL are eligible for WGS at diagnosis via the National Health Service, Genomic Medicine Service (NHS GMS). Methods Sixty-two patients, presenting with ALL at either Great Ormond Street Hospital or Bristol Children's Hospital, had short-read WGS via the NHS GMS performed concurrently with standard of care (SOC) targeted HTS to identify IG/TCR rearrangements. A bespoke bioinformatic pipeline was developed to identify IG/TCR rearrangements in the WGS samples. SOC targeted HTS analysis was performed using the open source Vidjil bioinformatic software. A cut-off of 5% (clone proportion of total clonotypes) was used to define an acceptable disease-specific clone for monitoring. Results All 62 samples had a greater number of rearrangements detected by WGS than targeted HTS. The mean number of clones &amp;gt;5% detected was 2.7 by targeted HTS and 3.1 by WGS. For rearrangements identified by targeted HTS at a 5% cut-off, 97% (185 out of 190) were identified by WGS. All targets were identified by WGS using a 10% cut-off (Table 1).Despite the lower depth of coverage of the WGS assay, it retrieved 89% of sequences seen in SOC down to a level of 1%, as well as additional re-arrangements not seen by the SOC assay (Figure 1). As well as additional rearrangements being discovered by WGS, we observed novel rearrangements (dd3/ja rearrangements) not previously identified using a targeted approach. To establish if the additional sequences identified by WGS were clinically applicable as MRD targets, we designed allele specific oligonucleotides for a representative 32 rearrangements for testing by qPCR. Over half (n=16) were able to be used to track MRD in follow-up samples. New primer/probe sets were designed for the novel dd3/ja rearrangements identified by WGS, these were also tested by qPCR and performed well enough to be clinically applicable for MRD monitoring. Conclusions WGS approach outperformed targeted HTS, identifying more IG/TCR rearrangements suitable for molecular MRD monitoring in all patients. This is of particularly benefit in cases where targeted HTS only identifies 1 or 2 potential MRD targets. WGS also has the advantage of identifying other key gene rearrangements (BCR-ABL, KMT2A, SIL-TAL, IKZF1) that make excellent targets for MRD monitoring. Beyond this, WGS delivers all diagnostic information, germline and additional somatic findings that aid diagnosis. For ALL, in one test WGS can now replace all molecular diagnostics. In England all patients with leukemia are eligible for centralised WGS. With continued improvements in turnaround times, and reducing costs, we propose that WGS should be the new diagnostic standard for pediatric ALL

BIOM-61. EXPLORING HOMOLOGOUS RECOMBINATION DEFICIENCY IN PEDIATRIC HIGH-GRADE GLIOMAS

Abstract Homologous recombination deficiency (HRD) has been extensively studied in breast cancer and ovarian cancer given its correlation with sensitivity to Poly (ADP-ribose) polymerases (PARP) inhibitors. In these cancers, HRD is most frequently caused by biallelic inactivation of BRCA1/2 and tumors often exhibit ‘genomic scars’ such as elevated HRD.sum score and specific mutational signatures, including specific single nucleotide variant (SNV), small insertion/deletion (Indel), and structural variant (SV) signatures. Despite its clinical relevance, HRD has not been systematically studied in pediatric cancers and its therapeutic predictive value is unknown. Here, we evaluated HRD in 255 tumor-normal paired whole-genome-sequenced (WGS) samples of pediatric high-grade glioma (pHGG) and 23 tumor-normal paired WGS samples of breast cancer (BC) as comparison. We found that 9% of the pHGG samples have HRD.sum score greater than 40, an indicator of HRD in several adult cancers. However, while we identified HRD-related SNV signature SBS3 and SV signature RS5 in some of the pHGG samples, HRD-related Indel signature ID6 was not identified. We also evaluated two composite scores, namely, HRDetect and CHORD, on our samples. While virtually all BC samples with BRCA1/2 loss were predicted to be HRD by both scores, none of the pHGG samples was predicted to be HRD by either score. We profiled somatic and germline variants from over 500 DNA damage response genes in the pHGG samples and identified TP53 as the most frequently mutated genes with biallelic loss, whereas biallelic loss of BRCA1/2 was not found. Overall, our study suggests that HRD exists in a fraction of pHGG samples, but is driven by inactivation of genes other than BRCA1/2, resulting in a different set of genomic scars compared to those in BCs. Functional analysis of sensitivity to PARP inhibitor and related DNA damage inhibitors is ongoing to evaluate potential therapeutic relevance of these genomic scars.

Landscape of enhancer disruption and functional screen in melanoma cells

BackgroundThe high mutation rate throughout the entire melanoma genome presents a major challenge in stratifying true driver events from the background mutations. Numerous recurrent non-coding alterations, such as those in enhancers, can shape tumor evolution, thereby emphasizing the importance in systematically deciphering enhancer disruptions in melanoma.ResultsHere, we leveraged 297 melanoma whole-genome sequencing samples to prioritize highly recurrent regions. By performing a genome-scale CRISPR interference (CRISPRi) screen on highly recurrent region-associated enhancers in melanoma cells, we identified 66 significant hits which could have tumor-suppressive roles. These functional enhancers show unique mutational patterns independent of classical significantly mutated genes in melanoma. Target gene analysis for the essential enhancers reveal many known and hidden mechanisms underlying melanoma growth. Utilizing extensive functional validation experiments, we demonstrate that a super enhancer element could modulate melanoma cell proliferation by targeting MEF2A, and another distal enhancer is able to sustain PTEN tumor-suppressive potential via long-range interactions.ConclusionsOur study establishes a catalogue of crucial enhancers and their target genes in melanoma growth and progression, and illuminates the identification of novel mechanisms of dysregulation for melanoma driver genes and new therapeutic targeting strategies.

The landscape of human SVA retrotransposons.

SINE-VNTR-Alu (SVA) retrotransposons are evolutionarily young and still-active transposable elements (TEs) in the human genome. Several pathogenic SVA insertions have been identified that directly mutate host genes to cause neurodegenerative and other types of diseases. However, due to their sequence heterogeneity and complex structures as well as limitations in sequencing techniques and analysis, SVA insertions have been less well studied compared to other mobile element insertions. Here, we identified polymorphic SVA insertions from 3646 whole-genome sequencing (WGS) samples of>150 diverse populations and constructed a polymorphic SVA insertion reference catalog. Using 20 long-read samples, we also assembled reference and polymorphic SVA sequences and characterized the internal hexamer/variable-number-tandem-repeat (VNTR) expansions as well as differing SVA activity for SVA subfamilies and human populations. In addition, we developed a module to annotate both reference and polymorphic SVA copies. By characterizing the landscape of both reference and polymorphic SVA retrotransposons, our study enables more accurate genotyping of these elements and facilitate the discovery of pathogenic SVA insertions.

REVIEW OF THE INTRODUCTION OF WHOLE GENOME SEQUENCING AS NHS STANDARD OF CARE FOR BRAIN TUMOUR PATIENTS AT QUEEN ELIZABETH HOSPITAL BIRMINGHAM, UK

Abstract AIMS Describe delays in the NHS pathway from initial consent for whole genome sequencing (WGS) to delivery of the final WGS report (WGS-R) to the treating oncologist. Review number of patients with mutations that inform, diagnosis, prognosis or treatment. METHOD Prospective audit of time to key stages along WGS pathway and review of final WGS-R. RESULTS WGS pathway and process: Data snapshot at 24/02/2023 showed 96 patients consented for WGS: 12/96 awaiting surgery; 41/96 awaiting GTAB discussion; 1/96 WGS sample used for histopathological diagnosis, 1/96 ineligible. 41/96 WGS results discussed at a GTAB meeting; 39/44 with complete WGS-R sent to the treating oncologist, including discussion at the local neuro-oncology multi-disciplinary team meeting. Median time from consent to final WGS-R was 225 days (range 145-567). WGS results: 4 patients with glioblastoma (GBM) have RB1 variant, associated with improved progression-free and overall survival (Dono et al 2021). 10 patients had clinically actionable mutations with recommended trials or compassionate-use drugs. 1/10 patient with GBM and High Tumour Mutational Burden who completed standard of care (SOC) treatment has commenced anti-PD1 immunotherapy (Nivolumab; Goodman et al 2017). 5/10 patients are currently on SOC treatment, 3/10 had deteriorated and 1/10 were deceased at time of WGS-R. Excellent concordance of WGS data with SOC genetic/immunohistochemistry testing. No germline mutations identified. CONCLUSIONS Nearly a third of patients had genetic variants that may have the potential to inform future clinical management. Further investment in the WGS pathway is required to deliver timely results to allow application of precision medicine for brain cancer patients.

Comparison of the Whole-Genome Sequence of the African Swine Fever Virus from a Mongolian Wild Boar with Genotype II Viruses from Asia and Europe.

African swine fever (ASF) is a highly contagious and severe viral hemorrhagic disease in domestic and wild pigs. ASF seriously affects the global swine industry as the mortality rate can reach 100% with highly virulent strains. In 2007, ASF was introduced into the Caucasus and spread to Russia and later into other European and Asian countries. This study reported the first whole-genome sequence (WGS) of the ASF virus (ASFV) that was detected in a Mongolian wild boar. This sequence was then compared to other WGS samples from Asia and Europe. Results show that the ASFV Genotype II from Mongolia is similar to the Asian Genotype II WGS. However, there were three nucleotide differences found between the Asian and European genome sequences, two of which were non-synonymous. It was also observed that the European Genotype II ASFV WGS was more diverse than that of the Asian counterparts. The study demonstrates that the ASFV Genotype II variants found in wild boars and domestic pigs are highly similar, suggesting these animals might have had direct or indirect contact, potentially through outdoor animal breeding. In conclusion, this study provides a WGS and mutation spectrum of the ASFV Genotype II WGS in Asia and Europe and thus provides important insights into the origin and spread of ASFV in Mongolia.

Genome-wide scan for runs of homozygosity in South American Camelids

BackgroundAlpaca (Vicugna pacos), llama (Lama glama), vicugna (Vicugna vicugna) and guanaco (Lama guanicoe), are the camelid species distributed over the Andean high-altitude grasslands, the Altiplano, and the Patagonian arid steppes. Despite the wide interest on these animals, most of the loci under selection are still unknown. Using whole-genome sequencing (WGS) data we investigated the occurrence and the distribution of Runs Of Homozygosity (ROHs) across the South American Camelids (SACs) genome to identify the genetic relationship between the four species and the potential signatures of selection.ResultsA total of 37 WGS samples covering the four species was included in the final analysis. The multi-dimensional scaling approach showed a clear separation between the four species; however, admixture analysis suggested a strong genetic introgression from vicugna and llama to alpaca. Conversely, very low genetic admixture of the guanaco with the other SACs was found. The four species did not show significant differences in the number, length of ROHs (100-500 kb) and genomic inbreeding values. Longer ROHs (> 500 kb) were found almost exclusively in alpaca. Seven overlapping ROHs were shared by alpacas, encompassing nine loci (FGF5, LOC107034918, PRDM8, ANTXR2, LOC102534792, BSN, LOC116284892, DAG1 and RIC8B) while nine overlapping ROHs were found in llama with twenty-five loci annotated (ERC2, FZD9, BAZ1B, BCL7B, LOC116284208, TBL2, MLXIPL, PHF20, TRNAD-AUC, LOC116284365, RBM39, ARFGEF2, DCAF5, EXD2, HSPB11, LRRC42, LDLRAD1, TMEM59, LOC107033213, TCEANC2, LOC102545169, LOC116278408, SMIM15, NDUFAF2 and RCOR1). Four overlapping ROHs, with three annotated loci (DLG1, KAT6B and PDE4D) and three overlapping ROHs, with seven annotated genes (ATP6V1E1, BCL2L13, LOC116276952, BID, KAT6B, LOC116282667 and LOC107034552), were detected for vicugna and guanaco, respectively.ConclusionsThe signatures of selection revealed genomic areas potentially selected for production traits as well as for natural adaptation to harsh environment. Alpaca and llama hint a selection driven by environment as well as by farming purpose while vicugna and guanaco showed selection signals for adaptation to harsh environment. Interesting, signatures of selection on KAT6B gene were identified for both vicugna and guanaco, suggesting a positive effect on wild populations fitness. Such information may be of interest to further ecological and animal production studies.

Chromosome-aware phylogenomics of Assassin Bugs (Hemiptera: Reduvioidea) elucidates ancient gene conflict.

Though the phylogenetic signal of loci on sex chromosomes can differ from those on autosomes, chromosomal-level genome assemblies for non-vertebrates are still relatively scarce and conservation of chromosomal gene content across deep phylogenetic scales has therefore remained largely unexplored. We here assemble a uniquely large and diverse set of samples (17 Anchored Hybrid Enrichment [AHE], 24 RNA-Seq, and 70 whole-genome sequencing [WGS] samples of variable depth) for the medically important assassin bugs (Reduvioidea). We assess the performance of genes based on multiple features (e.g., nucleotide vs. amino acid, nuclear vs. mitochondrial, and autosomal vs. X chromosomal) and employ different methods (concatenation and coalescence analyses) to reconstruct the unresolved phylogeny of this diverse (∼7,000 spp.) and old (>180 MYA) group. Our results show that genes on the X chromosome are more likely to have discordant phylogenies than those on autosomes. We find that the X chromosome conflict is driven by high gene substitution rates that impact accuracy of phylogenetic inference. However, gene tree clustering showed strong conflict even after discounting variable third codon positions. Alternative topologies were not particularly enriched for sex chromosome loci, but spread across the genome. We conclude that binning genes to autosomal or sex chromosomes may result in a more accurate picture of the complex evolutionary history of a clade.

ADSP Whole Genome Sequencing (WGS) Release 3 data update from Genome Center for Alzheimer’s Disease

AbstractBackgroundThe Genome Center for Alzheimer’s Disease (GCAD) coordinates the integration and meta‐analysis of all available Alzheimer’s disease (AD) relevant whole genome sequencing (WGS) data with the goal of identifying AD risk or protective genetic variants and eventual therapeutic targets. The WGS datasets are generated via the collaboration of scientists from the Alzheimer’s Disease Sequencing Project (ADSP) and GCAD. With the vision to minimize data heterogeneity, introduced by different sequencing protocols and machines, GCAD processes all samples using identical pipelines and performs quality assurance (QA) checks.MethodsRaw sequencing data (FASTQs or BAMs) were aligned to GRCh38/hg38 by BWA, and variant calling and joint genotyping were done by GATK. Furthermore, Smoove, Manta and Streka were applied to generate structural variant (SV) calls per sample. QA checks including sex, contamination and genotype concordance as well as the ADSP QC protocol were performed to evaluate the quality of samples and variants. To facilitate the access and usage of the big joint‐genotyped VCF files, we introduced a compact version for storing variant info and sample genotypes only.ResultsWe dropped 235 (1.3%) samples of poor coverage (&lt;20x) or that failed QA checks, and we flagged 173 (1.0%) samples that were of borderline quality. As a result, the dataset (ADSP Release 3, 2021) includes 16,905 genomes from 17 diverse cohorts with 3 major ethnicities: 10,651 Non‐Hispanic Whites, 3,212 Hispanics and 2,874 African Americans. Data are deeply sequenced (average genome coverage: &gt;30x). All samples’ CRAMs, gVCFs from GATK, and VCFs from the three SV callers were deposited into NIAGADS Data Sharing Service (DSS) (https://dss.niagads.org/) for public distribution. In addition, joint‐genotype VCFs are available in both compact and QC versions. This joint‐genotype VCF contains &gt;206M bi‐allelic single‐nucleotide variants, 16M bi‐allelic indels and 28M multi‐allelic variants, with 96% of variants remaining after stringent QC.ConclusionThe ADSP and GCAD generate high quality genotype calls and SV calls. Currently the project is processing ∼37,000 WGS samples sequenced primarily through the ADSP Follow‐Up Study, which will contain a more ancestrally diverse set of populations. We anticipate this 2022 release will continue to benefit the research community studying AD genetics.

Abstract 3330: Development of a genome-wide multiomic atlas of early-stage lung cancer enables identification of novel methylation biomarkers for disease detection beyond TCGA

Abstract Lung cancer is the leading cause of cancer mortality, mainly due to diagnosis at advanced stages. Early detection reduces mortality and screening by CT is recommended for a high-risk population, however, uptake is low. Biomarkers for early-stage detection may enhance screening. Liquid biopsy and blood circulating tumor DNA (ctDNA) are established plasma surrogates of tumor tissue but their use in early detection remains challenging due to the high noise level of current techniques. We constructed a novel multiomic atlas through whole genome sequencing (WGS) to map all genetic and epigenetic changes associated with early-stage lung cancer as a tool to identify markers. Biospecimens from 48 early-stage lung cancer cases (58% stage I) and 29 cancer-free controls, all high-risk by USPSTF, were acquired from academic (UBC, Vanderbilt, Cleveland Clinic) and commercial biobanks. Cases had tumor and normal lung tissue, whole blood (WB) and plasma samples. Controls had WB and plasma samples. EpiCheck sequencing (ECS) which combines methylation-sensitive restriction endonuclease digestion to detect differential methylation with WGS for multiomic analyses was performed on each sample. Extracted DNA was digested, underwent standard library preparation and sequenced at an average depth of 600x for cfDNA and 80x for tissue and WB. A subset also underwent WG bisulfite sequencing (BS) and standard WGS for comparison. Data analysis was performed using customized software. ECS outperformed BS. Mapping rate was 99.6%, 99.7% and 85.7% and unique mapping rate was 94.1%, 94.3% and 81.4% for WGS, ECS, and BS samples, respectively. Copy number integrity showed Pearson correlations of 0.9 for ECS and 0.67 for BS. Somatic mutation analysis in tissue identified a subset of cases with relatively high ctDNA shedding in plasma that were associated with larger tumors, older age and squamous cell carcinoma histology. This subset was further used to identify tumor derived plasma-based markers and assess fragmentation with high confidence. Shorter ctDNA fragments were observed in some, but not all cases. Additional analysis of WB and plasma identified host derived blood-based markers using methylation and copy number. This orthogonal approach enabled detection of small, low shedding tumors. A discovery panel of 87 markers showed 100% sensitivity and 97% specificity to discriminate cases vs controls. Only 11% of these markers are represented in the TCGA data. ECS generates high integrity sequencing data superior to BS, enabling genome-wide multiomic analyses (methylation, mutation, copy number, fragmentomics), with methylation signatures beyond the scope of TCGA. Results, which require validation, underscore the potential of the EpiCheck lung cancer atlas as a development platform of novel blood biomarkers for early-stage lung cancer detection. Citation Format: Revital Knirsh, Stephen Lam, Anna McGuire, Peter J. Mazzone, Stephen Deppen, Eric Grogan, Fabien Maldonado, Orna Savin, Shacade Danan, Sarah Zaouch, Nimrod Axelrad, Dvir Netanely, Aharona Shuali, Catherine A. Schnabel, Adam Wasserstrom, Danny Frumkin. Development of a genome-wide multiomic atlas of early-stage lung cancer enables identification of novel methylation biomarkers for disease detection beyond TCGA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3330.

Composition and function of the Galapagos penguin gut microbiome vary with age, location, and a putative bacterial pathogen

Microbial colonization plays a direct role in host health. Understanding the ecology of the resident microbial community for a given host species is thus an important step for detecting population vulnerabilities like disease. However, the idea of integrating microbiome research into conservation is still relatively new, and wild birds have received less attention in this field than mammals or domesticated animals. Here we examine the composition and function of the gut microbiome of the endangered Galapagos penguin (Spheniscus mendiculus) with the goals of characterizing the normal microbial community and resistome, identifying likely pathogens, and testing hypotheses of structuring forces for this community based on demographics, location, and infection status. We collected fecal samples from wild penguins in 2018 and performed 16S rRNA gene sequencing and whole genome sequencing (WGS) on extracted DNA. 16S sequencing revealed that the bacterial phyla Fusobacteria, Epsilonbacteraeota, Firmicutes, and Proteobacteria dominate the community. Functional pathways were computed from WGS data, showing genetic functional potential primarily focused on metabolism—amino acid metabolism, carbohydrate metabolism, and energy metabolism are the most well-represented functional groups. WGS samples were each screened for antimicrobial resistance, characterizing a resistome made up of nine antibiotic resistance genes. Samples were screened for potential enteric pathogens using virulence factors as indicators; Clostridium perfringens was revealed as a likely pathogen. Overall, three factors appear to be shaping the alpha and beta diversity of the microbial community: penguin developmental stage, sampling location, and C. perfringens. We found that juvenile penguins have significantly lower alpha diversity than adults based on three metrics, as well as significantly different beta diversity. Location effects are minimal, but one site has significantly lower Shannon diversity than the other primary sites. Finally, when samples were grouped by C. perfringens virulence factors, we found dramatic changes in beta diversity based on operational taxonomic units, protein families, and functional pathways. This study provides a baseline microbiome for an endangered species, implicates both penguin age and the presence of a potential bacterial pathogen as primary factors associated with microbial community variance, and reveals widespread antibiotic resistance genes across the population.

Abstract P198: Associations Between Plasma Proteome and Leukocyte Mitochondrial DNA Copy Number: The Atherosclerosis Risk in Communities Study (ARIC)

Introduction: The number of copies of the mitochondrial genome, termed mitochondrial DNA copy number (MTCN), is a proxy for mitochondrial function and varies by cell. Critically, lower MTCN has been associated with aging and chronic diseases such as dementia and cancer. We conducted a large-scale proteomics analysis of leukocyte MTCN (L-MTCN) to elucidate pathways modulated by L-MTCN that may be involved in diseases of aging. Hypothesis: We hypothesize that L-MTCN is significantly associated to plasma proteins that have been implicated in biological pathways of L-MTCN and related diseases. Methods: Based on whole genome sequencing (WGS) data measured in samples collected in ARIC visits 1, 2, and 3 (i.e., baseline for the current study), L-MTCN was calculated as (2 * coverage of the mitochondrial genome) / coverage of the autosome, using the median coverage of 1000 base pair bins overlapping the mitochondrial genome (16 total) and the median coverage of ~142,000 higher quality autosomal bins. Only samples with read lengths of 150 or 151 were included in subsequent analyses, and L-MTCN estimates were inverse normalized within read length group and WGS group before being merged together. This normalized L-MTCN was then analyzed as a predictor for an association with 4,870 plasma proteins measured by SOMAscan v4 in ARIC visit 3 in race-specific generalized linear models, adjusting for the following covariates at baseline: WGS sample visit, age, gender, field center, smoking status, and a measure of kidney function. Bonferroni correction was applied to account for the number of proteins tested in 5,007 White participants (p≤1x10 -5 ). Associations were tested for replication in 1,178 Black participants and considered significant if protein estimates had the same direction of association as Whites at p-value ≤ 2.2x10 -4 . Results: Among White participants, 230 proteins were significantly associated with L-MTCN. Of these proteins S100A9, GP1Bα, EGFR, THBS2, and FCGR3B were significantly replicated among Black participants. Strongest associations with L-MTCN were seen with GP1Bα (Estimate: 0.121) and S100A9 (Estimate: -0.191). Biologically S100A9 is a calgranulin and together with S100A8 forms calprotectin, which have roles mediating inflammatory processes. GP1Bα helps mediate platelet activation and forms the glycoprotein Ib-IX-V complex which acts as a platelet surface receptor for von Willebrand factor facilitating platelet adhesion. Conclusion: This large proteomics analysis identified 230 plasma proteins significantly associated with L-MTCN. Some of the proteins have been connected to inflammatory diseases, cardiovascular disease, and cancer in the literature. These results provide insight into proteins involved in biological pathways related to L-MTCN.

The repertoire of copy number alteration signatures in human cancer.

Copy number alterations (CNAs) are a predominant source of genetic alterations in human cancer and play an important role in cancer progression. However comprehensive understanding of the mutational processes and signatures of CNA is still lacking. Here we developed a mechanism-agnostic method to categorize CNA based on various fragment properties, which reflect the consequences of mutagenic processes and can be extracted from different types of data, including whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) array. The 14 signatures of CNA have been extracted from 2778 pan-cancer analysis of whole genomes WGS samples, and further validated with 10851 the cancer genome atlas SNP array dataset. Novel patterns of CNA have been revealed through this study. The activities of some CNA signatures consistently predict cancer patients' prognosis. This study provides a repertoire for understanding the signatures of CNA in cancer, with potential implications for cancer prognosis, evolution and etiology.