Coverage Whole Genome Sequencing Research Articles

Variants in LPA are associated with Familial Hypercholesterolaemia: whole genome sequencing analysis in the 100,000 Genomes Project.

Familial Hypercholesterolaemia (FH) is an inherited disease of high LDL-cholesterol (LDL-C) caused by defects in LDLR, APOB, APOE and PCSK9 genes. A pathogenic variant cannot be found in ∼60% of clinical FH patients. Using whole genome sequencing (WGS) we examined genetic determinants of FH. WGS data generated by the 100,000 Genomes Project (100KGP) included 536 FH patients diagnosed using the FH Simon Broome criteria. Rare variants in known FH genes were analysed. Genome-wide association study (GWAS) between 443 FH variant-negative unrelated FH cases and 77,275 control participants of the 100KGP was run using high coverage WGS data. Polygenic risk scores for LDL-C (LDL PRS) and lipoprotein(a) (Lp(a) PRS) were computed. An FH-causing variant was found in 17.4% of FH cases. GWAS identified the LPA gene locus being significantly associated (p<1x10-8). FH variant-negative participants had higher LDL and Lp(a) PRSs in comparison to the controls (p<1.0×10-16 and p<4.09×10-6, respectively). Similar associations were found in the monogenic FH with both LDL and Lp(a) PRSs being higher than in controls (p<4.03×10-4 and p<3.01x10-3, respectively). High LDL PRS was observed in 36.4% of FH variant-negative cases, whereas high Lp(a) PRS in 18.5%, with 7.0% having both high LDL and Lp(a) PRSs. This genome-wide analysis of monogenic and polygenic FH causes confirms a complex and heterogenous architecture of hypercholesterolaemia, with the LPA gene playing a significant role. Both Lp(a) and LDL-C should be measured for precision FH diagnosis. Specific therapies to lower Lp(a) should be targeted to those who will benefit most.

Abstract 1764: Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions

Abstract Background Acute myeloid leukemia with complex karyotype (CKAML) does frequently show del(5q), deletion of chromosome 5q. Previous work in MDS and AML showed that CKAML form clusters distinguishing del(5q) cases with commonly retained regions (CRRs) in the telomeric (≥5q34) and centromeric (≤5q14.2) ends from cases with loss of the telomeric and/or centromeric regions. Interestingly, del(5q) patients with CRRs were shown to have less genomic lesions and were associated with a better prognosis than other 5q-deleted CKAML cases. Aim: Combining the advantages of two different long read sequencing technologies, Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), we aimed to more comprehensively characterize the molecular landscape of CKAML with del(5q). Our integrative workflow enabled an in-depth characterization of copy number variations (CNV), genomic rearrangements, transcriptomic and epigenomic changes. Methods: Low coverage whole genome sequencing (WGS) using ONT was performed on 209 patients with CKAML to identify del(5q) cases for further in-depth analysis. For 10 selected cases with known CRR status, genomic deep sequencing using PacBio Revio and ONT PromethION was performed, as well as PacBio Revio MAS-seq for fusion transcript detection. Results: A mean sequencing depth of 3-fold (range 0.4-17.3-fold) was obtained by low coverage WGS of the 209 CKAML cases. With a resolution of 0.1 Mbp n=134 del(5q) cases were identified in our CKAML cohort (64%) and commonly deleted regions on 5q could be resolved at 5q21.2 to 5q23.3 and 5q33.3 to 5q35 (Fisher test, FDR Benjamini-Hochberg, p&amp;lt;10−30). These regions separated del(5q) in n=52 cases showing CRR retention and n=82 showing partially no CRR, (either the centromeric or telomeric regions, n=74, or of both regions, n=8 involved). Subsequent co-occurrence analysis provided evidence of an association between the loss of telomeric 5q region with a 3p12-25 deletion. In-depth analysis (30-50 fold) of ten distinct del(5q) cases by PacBio and ONT revealed additional insights into the complex clonal composition of del(5q) cases. For example, one case showed two distinct subclones with 5q14.2-5q35.1 deletion and with translocation t(3;5)(p14;q14) respectively. Moreover, we could demonstrate that 5q-aberrations are far more complex than initially thought showing, e.g. inverted terminal duplications that resulted in fusion transcripts involving PDE4D and leading to its deregulated expression. Conclusion: Using our long read deep sequencing approach we could further delineate the structural complexity of del(5q) CKAML. While gene expression profiling, methylation analysis and correlation of findings with clinical outcome are ongoing and will be presented at the meeting, our results help to further pinpoint the biology underlying del(5q) and to further refine this heterogeneous CKAML subgroup. Citation Format: Felix Schick, Ekaterina Slonova, Eric Straeng, Sarah Huet, Maurizio Raso, Marlon Tilgner, Frederik Damm, Pierre Sujobert, Olga Blau, Lars Bullinger, Anna Dolnik. Long read sequencing allows comprehensive molecular profiling of complex karyotype acute myeloid leukemia (CKAML) with 5q deletions [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 1764.

Identifying risk variants for embryo aneuploidy using ultra-low coverage whole-genome sequencing from preimplantation genetic testing

Aneuploidy frequently arises during human meiosis and is the primary cause of early miscarriage and invitro fertilization (IVF) failure. Individuals undergoing IVF exhibit significant variability in aneuploidy rates, although the exact genetic causes of the variability in aneuploid egg production remain unclear. Preimplantation genetic testing for aneuploidy (PGT-A) using next-generation sequencing is a standard test for identifying and selecting IVF-derived euploid embryos. The wealth of embryo aneuploidy data and ultra-low coverage whole-genome sequencing (ulc-WGS) data from PGT-A have the potential to discover variants in parental genomes that are associated with aneuploidy risk in their embryos. Using ulc-WGS data from ∼10,000 PGT-A biopsies, we imputed genotype likelihoods of genetic variants in embryo genomes. We then used the imputed variants and embryo aneuploidy calls to perform a genome-wide association study of aneuploidy incidence. Finally, we carried out functional evaluation of the identified candidate gene in a mouse oocyte system. We identified one locus on chromosome 3 that is significantly associated with meiotic aneuploidy risk. One candidate gene, CCDC66, encompassed by this locus, is involved in chromosome segregation during meiosis. Using mouse oocytes, we showed that CCDC66 regulates meiotic progression and chromosome segregation fidelity, especially in older mice. Our work extended the research utility of PGT-A ulc-WGS data by allowing robust association testing and improved the understanding of the genetic contribution to maternal meiotic aneuploidy risk. Importantly, we introduce a generalizable method that has potential to be leveraged for similar association studies that use ulc-WGS data.

Assessment and validation of enrichment and target capture approaches to improve Mycobacterium tuberculosis WGS from direct patient samples.

Within-host Mycobacterium tuberculosis (Mtb) diversity may detect antibiotic resistance or predict tuberculosis treatment failure and is best captured through sequencing directly from sputum. Here, we compared three sample pre-processing steps for DNA decontamination and studied the yield of a new target enrichment protocol for optimal whole-genome sequencing (WGS) from direct patient samples. Mtb-positive NALC-NaOH-treated patient sputum sediments were pooled, and heat inactivated, split in replicates, and treated by either a wash, DNase I, or benzonase digestion. Levels of contaminating host DNA and target Mtb DNA were assessed by quantitative PCR (qPCR), followed by WGS with and without custom dsDNA target enrichment. The pre-treatment sample has a high host-to-target ratio of DNA (6,168 ± 1,638 host copies/ng to 212.3 ± 59.4 Mtb copies/ng) that significantly decreased with all three treatments. Benzonase treatment resulted in the highest enrichment of Mtb DNA at 100-fold compared with control (3,422 ± 2,162 host copies/ng to 11,721 ± 7,096 Mtb copies/ng). The custom dsDNA probe panel successfully enriched libraries from as little as 0.45 pg of Mtb DNA (100 genome copies). Applied to direct sputum the dsDNA target enrichment panel increased the percent of sequencing reads mapping to the Mtb target for all three pre-processing methods. Comparing the results of the benzonase sample sequenced both with and without enrichment, the percent of sequencing reads mapping to the Mtb increased to 90.95% from 1.18%. We demonstrate a low limit of detection for a new custom dsDNA Mtb target enrichment panel that has a favorable cost profile. The results also demonstrate that pre-processing to remove contaminating extracellular DNA prior to cell lysis and DNA extraction improves the host-to-Mtb DNA ratio but is not adequate to support average coverage WGS without target capture.

Construction of high coverage whole-genome sequencing libraries from single colon crypts without DNA extraction or whole-genome amplification

ObjectiveComprehensive and reliable genome-wide variant analysis of a small number of cells has been challenging due to genome coverage bias, PCR over-cycling, and the requirement of expensive technologies. To comprehensively identify genome alterations in single colon crypts that reflect genome heterogeneity of stem cells, we developed a method to construct whole-genome sequencing libraries from single colon crypts without DNA extraction, whole-genome amplification, or increased PCR enrichment cycles.ResultsWe present post-alignment statistics of 81 single-crypts (each contains four- to eight-fold less DNA than the requirement of conventional methods) and 16 bulk-tissue libraries to demonstrate the consistent success in obtaining reliable coverage, both in depth (≥ 30X) and breadth (≥ 92% of the genome covered at ≥ 10X depth), of the human genome. These single-crypt libraries are of comparable quality as libraries generated with the conventional method using high quality and quantities of purified DNA. Conceivably, our method can be applied to small biopsy samples from many tissues and can be combined with single cell targeted sequencing to comprehensively profile cancer genomes and their evolution. The broad potential application of this method offers expanded possibilities in cost-effectively examining genome heterogeneity in small numbers of cells at high resolution.

Whole-genome sequencing reveals a complex African population demographic history and signatures of local adaptation.

We conduct high coverage (>30×) whole-genome sequencing of 180 individuals from 12 indigenous African populations. We identify millions of unreported variants, many predicted to be functionally important. We observe that the ancestors of southern African San and central African rainforest hunter-gatherers (RHG) diverged from other populations >200 kya and maintained a large effective population size. We observe evidence for ancient population structure in Africa and for multiple introgression events from "ghost" populations with highly diverged genetic lineages. Although currently geographically isolated, we observe evidence for gene flow between eastern and southern Khoesan-speaking hunter-gatherer populations lasting until ∼12kya. We identify signatures of local adaptation for traits related to skin color, immune response, height, and metabolic processes. We identify a positively selected variant in the lightly pigmented San that influences pigmentation invitro by regulating the enhancer activity and gene expression of PDPK1.

Rapid and Reproducible Karyotyping with Nanopore Sequencing in AML Patients

Introduction: We evaluated low coverage whole genome sequencing (WGS) of acute myeloid leukemia (AML) patients using long read Oxford Nanopore Technology (ONTseq) for karyotyping and compared the results and the frequency of patients with myelodysplasia-related cytogenetic abnormalities according to previous and current AML classifications with conventional cytogenetics (CG). Methods: Diagnostic samples from peripheral blood or bone marrow of 100 AML patients were sequenced in 3 medical centers on a GridION sequencer using SQK-LSK109 kits according to the manufacturer's instructions, and analyzed by CG. Ten samples were sequenced independently in two different laboratories. Fifty samples were sequenced prospectively from newly diagnosed de novo AML patients. Sequencing reads were analyzed by an in-house bioinformatics pipeline based on publicly available tools such as nglmr aligner, samtools, igv_tools, R package ACE, the CyDAS webserver, and custom scripts for transforming ACE copy number models to karyotype formulas. CNVs from ONTseq data were mapped to 290 chromosome bands to allow comparison with CG. We compared the following cytogenetic categories: Complex karyotype (cKT) ≥ 3 chromosomal abnormalities; monosomal karyotype (mKT) as defined by Breems et al.; karyotype with myelodysplasia-related cytogenetic abnormalities that defines MRC-AML according WHO 2016 classification (MRC-KT), and ICC 2022 classification (MRCA-KT). Results: ONTseq of 100 AML patients with a 4.4-fold genome coverage (range: 2.1-6.2) allowed us to resolve CNVs at a high resolution of ≥0.1 mega-basepairs (Mbp). Analysis of the ten duplicate samples established a high reproducibility of ONTseq. 110 CNVs were detected by both laboratories at identical chromosome positions and similar length (R2=0.999). Only 8 CNVs (7%) differed considerably in length between the replicates (median length difference 2 Mbp, range 0.4-16 Mbp). Of 100 patients with a median age of 65 years (range 18-97), ≥1 chromosome band was gained or lost in 69 patients according to ONTseq analysis, while CNVs or balanced translocations were found by cytogenetics in 72 patients. Five patients had a normal karyotype by ONTseq, but not by CG, while two patients had a normal karyotype by CG but not by ONTseq. Two of these 7 discrepant patients had translocations as their sole cytogenetic abnormality [t(8;21) and t(9;11)], which cannot be detected by low coverage genome ONTseq. Patients with chromosomal abnormalities had a median of 29 (range 1-140) and 30 (range 3-136) chromosomal bands with CNVs according to ONTseq and CG, respectively. Considering all patients, gains and losses at the level of chromosomal bands correlated very well (gains: R2=0.922; losses: R2=0.923) (Figure 1). All patients were then classified according the definitions for cKT, mKT, MRC-KT, and MRCA-KT (Table 1). Overall, patients were similarly classified by ONTseq and CG results (P<0.001). MRC and MRCA-KTs were consistently identified by ONTseq and CG in 57 patients. In 2 and 2 patients, MRC and MRCA-KTs, respectively, were identified by CG only. In 1 and 1 patient, MRC and MRCA-KT, respectively, was identified by ONTseq only. The largest discrepancies were found for mKTs due to the higher resolution of ONTseq, which more often identified subchromosomal instead of whole chromosome CNVs compared to CG, which were not considered for mKT. Fifty of the 100 patients were sequenced prospectively at time of diagnosis. The median age of these patients was 70 years (range 61-97) and all patients had de novo AML. At least one CNV was detected by ONTseq in 24 (48%) and by CG in 26 (52%) patients. cKT, mKT, MRC-KT, and MRCA-KT were detected by ONTseq in 12, 7, 17, and 17 patients and by CG in 14, 11, 17, and 17 patients, respectively. The median time for library preparation for prospectively analyzed patients was 3:45 hours, for ONTseq 24 hours and for bioinformatic analysis 6:30 hours. In this setting, clinical reports can be delivered on the fourth day of sample receipt. Conclusion: Low coverage WGS with ONT provides a highly reproducible method for rapid karyotyping with high resolution. The identification of cKT and myelodysplasia-related cytogenetic abnormalities correlated very well with conventional cytogenetics, even though balanced translocations cannot be detected by our ONTseq protocol. Low coverage WGS is a promising approach for diagnostic karyotyping in AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EPCO-47. ITCC-P4: GENOMIC PROFILING AND ANALYSES OF PEDIATRIC PATIENT TUMOR AND PATIENT-DERIVED XENOGRAFT (PDX) MODELS FOR HIGH THROUGHPUT IN VIVO TESTING

Abstract Advancements in state-of-the-art molecular profiling techniques has resulted in better understanding of pediatric cancers and their drivers. Conversely, it also became apparent that pediatric cancers are much more heterogeneous than previously thought. Many new types and subtypes of pediatric cancers have been identified with distinct molecular and clinical characteristics. However, for most newly recognized entities there is no specific treatment available yet. The ITCC-P4 consortium is a collaboration between many academic centers across Europe and several pharmaceutical companies involved in preclinical testing, with the overall aim to establish a sustainable platform of &amp;gt;400 molecularly well-characterized PDX models of high-risk pediatric cancers and to use them for in vivo testing of novel mechanism-of-action based treatments. Currently, 340 models are fully established, including 87 brain and 253 non-brain tumor models, together representing different tumor types both from primary (113) and relapsed (92)/metastatic disease (42). 252 of these models have been fully molecularly characterized, representing 18 pediatric cancer entities and 43 different subtypes. Using low coverage whole-genome and whole exome sequencing, somatic mutation calling, DNA copy number, transcriptome analysis and methylation profiling we have observed that the molecular profile of most PDX models closely mimics their original tumors. Clonal evolution of somatic variants was only observed in some PDX-tumor pairs or so between disease states. Somatic copy number variant analysis highlights specific alterations for instance MYB, MYC, MYCN, NTRK3, PTEN loss differently distributed between PDX-patient tumor pairs in high-grade gliomas. Overall, our results show that we have established &amp;gt;250 PDX models of solid pediatric cancers, that well represents the disease spectrum and that is currently being used for in vivo testing of standard of care drugs and targeted small molecules. Treatment responses will be directly linked to molecular data to identify potential biomarkers for prioritization or deprioritization of individual, patient-specific specific drugs.

Identification of RP1 as the genetic cause of retinitis pigmentosa in a multi-generational pedigree using Extremely Low-Coverage Whole Genome Sequencing (XLC-WGS)

Motivation.Next-generation sequencing (NGS) technologies are decisive for discovering disease-causing variants, although their cost limits their utility in a clinical setting. A cost-mitigating alternative is an extremely low coverage whole-genome sequencing (XLC-WGS). We investigated its use to identify causal variants within a multi-generational pedigree of individuals with retinitis pigmentosa (RP). Causing progressive vision loss, RP is a group of genetically heterogeneous eye disorders with approximately 60 known causal genes. ResultsWe performed XLC-WGS in seventeen members of this pedigree, including three individuals with a confirmed diagnosis of RP. Sequencing data were processed using Illumina's DRAGEN pipeline and filtered using Illumina's genotype quality score metric (GQX). The resulting variants were analyzed using Expert Variant Interpreter (eVai) from enGenome as a prioritization tool. A nonsense known mutation (c.1625C > G; p.Ser542*) in exon 4 of the RP1 gene emerged as the most likely causal variant. We identified two homozygous carriers of this variant among the three sequenced RP cases and three heterozygous individuals with sufficient coverage of the RP1 locus. Our data show the utility of combining pedigree information with XLC-WGS as a cost-effective approach to identify disease-causing variants.

Detection and monitoring of HBV-related hepatocellular carcinoma from plasma cfDNA fragmentation profiles

Most hepatocellular carcinomas (HCCs) are associated with hepatitis B virus infection (HBV) in China. Early detection of HCC can significantly improve prognosis but is not yet fully clinically feasible. This study aims to develop methods for detecting HCC and studying the carcinogenesis of HBV using plasma cell-free DNA (cfDNA) whole-genome sequencing (WGS) data. Low coverage WGS was performed for 452 participants, including healthy individuals, hepatitis B patients, cirrhosis patients, and HCC patients. Then the sequencing data were processed using various machine learning models based on cfDNA fragmentation profiles for cancer detection. Our best model achieved a sensitivity of 87.10% and a specificity of 88.37%, and it showed an increased sensitivity with higher BCLC stages of HCC. Overall, this study proves the potential of a non-invasive assay based on cfDNA fragmentation profiles for the detection and prognosis of HCC and provides preliminary data on the carcinogenic mechanism of HBV.

Birth and death in terminal complement pathway

The cytolytic activity of the membrane attack complex (MAC) is pivotal in the complement-mediated elimination of pathogens. Terminal complement pathway (TCP) genes encode the proteins that form the MAC. Although the TCP genes are well conserved within most vertebrate species, the early evolution of the TCP genes is poorly understood. Based on the comparative genomic analysis of the early evolutionary history of the TCP homologs, we evaluated four possible scenarios that could have given rise to the vertebrate TCP. Currently available genomic data support a scheme of complex sequential protein domain gains that may be responsible for the birth of the vertebrate C6 gene. The subsequent duplication and divergence of this vertebrate C6 gene formed the C7, C8α, C8β, and C9 genes. Compared to the widespread conservation of TCP components within vertebrates, we discovered that C9 has disintegrated in the genomes of galliform birds. Publicly available genome and transcriptome sequencing datasets of chicken from Illumina short read, PacBio long read, and Optical mapping technologies support the validity of the genome assembly at the C9 locus. In this study, we have generated a > 120X coverage whole-genome Chromium 10x linked-read sequencing dataset for the chicken and used it to verify the loss of the C9 gene in the chicken. We find multiple CR1 (chicken repeat 1) element insertions within and near the remnant exons of C9 in several galliform bird genomes. The reconstructed chronology of events shows that the CR1 insertions occurred after C9 gene loss in an early galliform ancestor. Loss of C9 in galliform birds, in contrast to conservation in other vertebrates, may have implications for host-pathogen interactions. Our study of C6 gene birth in an early vertebrate ancestor and C9 gene death in galliform birds provides insights into the evolution of the TCP.

Copy Number Variation of Circulating Tumor DNA (ctDNA) Detected Using NIPT in Neoadjuvant Chemotherapy-Treated Ovarian Cancer Patients.

Analysis of circulating tumor DNA (ctDNA) can be used to characterize and monitor cancers. Recently, non-invasive prenatal testing (NIPT) as a new next-generation sequencing (NGS)-based approach has been applied for detecting ctDNA. This study aimed to investigate the copy number variations (CNVs) utilizing the non-invasive prenatal testing in plasma ctDNA from ovarian cancer (OC) patients who were treated with neoadjuvant chemotherapy (NAC). The plasma samples of six patients, including stages II–IV, were collected during the pre- and post-NAC treatment that were divided into NAC-sensitive and NAC-resistant groups during the follow-up time. CNV analysis was performed using the NIPT via two methods “an open-source algorithm WISECONDORX and NextGENe software.” Results of these methods were compared in pre- and post-NAC of OC patients. Finally, bioinformatics tools were used for data mining from The Cancer Genome Atlas (TCGA) to investigate CNVs in OC patients. WISECONDORX analysis indicated fewer CNV changes on chromosomes before treatment in the NAC-sensitive rather than NAC-resistant patients. NextGENe data indicated that CNVs are not only observed in the coding genes but also in non-coding genes. CNVs in six genes were identified, including HSF1, TMEM249, MROH1, GSTT2B, ABR, and NOMO2, only in NAC-resistant patients. The comparison of these six genes in NAC-resistant patients with The Cancer Genome Atlas data illustrated that the total alteration frequency is amplification, and the highest incidence of the CNVs (≥35% based on TCGA data) is found in MROH1, TMEM249, and HSF1 genes on the chromosome (Chr) 8. Based on TCGA data, survival analysis showed a significant reduction in the overall survival among chemotherapy-resistant patients as well as a high expression level of these three genes compared to that of sensitive samples (all, p < 0.0001). The continued Chr8 study using WISECONDORX revealed CNV modifications in NAC-resistant patients prior to NAC therapy, but no CNV changes were observed in NAC-sensitive individuals. Our findings showed that low coverage whole-genome sequencing analysis used for NIPT could identify CNVs in ctDNA of OC patients before and after chemotherapy. These CNVs are different in NAC-sensitive and -resistant patients highlighting the potential application of this approach in cancer patient management.

Early detection of hepatocellular carcinoma using cfDNA signatures from cirrhotic patients with nodules.

4113 Background: Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death worldwide. Early detection of HCC patients is related to favorable survival. Nodules in liver cirrhosis (LC) are at higher risk of developing into HCC. There is an urgent clinical need for development of an accurate and affordable non-invasive method for early HCC diagnosis among LC patients with nodules. Methods: We have previously shown that the genome-wide HIFI (5-Hydroxymethylcytosine/motIf/ Fragmentation/nucleosome footprInt) method held a solid diagnostic value in differentiating HCC from LC. In this study, we leverage this knowledge to diagnose early HCC (BCLC 0/A) in cirrhotic nodules using copy number variation (CNV) to replace 5-Hydroxymethylcytosine to simplify NGS protocol and decrease cost. The updated method generates a score that reflects the presence of tumor-derived cfDNA in 10 ml blood via low coverage (2x) whole-genome sequencing (WGS). We applied it in a retrospective cohort (validation set 1, n = 171) and a prospective cohort (validation set 2, n = 156), both of which involved patients with newly diagnosed early stage HCC (BCLC 0/A) as well as individuals with cirrhotic nodules. Results: The updated method showed excellent performance for early HCC detection both in the validation set 1 (84 HCC and 87 cirrhotic nodules; AUC: 0.951, 82.1% sensitivity at 90.8% specificity) and validation set 2 (71 HCC and 85 cirrhotic nodules; AUC: 0.958, 81.7% sensitivity at 91.8% specificity) (Table). The AUC values for distinguishing early HCC from cirrhotic nodules could reach 0.951 (80.9% sensitivity at 91.0% specificity) and 0.947 (74.7% sensitivity at 92.1% specificity) among patients with AFP &lt; 400 μg/L and those with nodular size &lt; = 2 cm, respectively. More importantly, our model also maintained consistent performance in detecting very early stage HCC (BCLC 0) with AUC of 0.941 (74.7% sensitivity at 91.3% specificity). Conclusions: These findings provide an accurate, affordable, excellent clinical potential model integrating four cfDNA molecular signatures for detecting early stage HCC from cirrhotic nodules using low-coverage WGS of plasma cfDNA samples. [Table: see text]

Development and Characterization of 20 Genomic SSR Markers for Ornamental Cultivars of Weigela.

Weigela (Caprifoliaceae) is a genus of ornamental plants popular for its phenotypic variation and hardiness, that includes species hybridized to produce the commercially available cultivars. Despite its popularity, limited genetic resources exist for the genus. Twenty genomic simple sequence repeat (gSSR) markers distributed across the genome were developed using low coverage whole-genome sequencing data of Weigela Spilled Wine®. A cross-amplification evaluation with these 20 gSSR markers on a collection of 18 Weigela cultivars revealed a total of 111 unique alleles, including 36 private alleles. A diagrammatic key was constructed to identify cultivars using only six of the gSSR markers, demonstrating the newly developed gSSR markers are immediately useful for cultivar identification. Future uses could include breeding with marker-assisted selection, determining the history of hybridization of the current cultivated lines, aiding in the construction of genetic maps, and assessing the patterns of population genetic structure of Weigela spp.

Contribution of rare whole-genome sequencing variants to plasma protein levels and the missing heritability

Despite the success of genome-wide association studies, much of the genetic contribution to complex traits remains unexplained. Here, we analyse high coverage whole-genome sequencing data, to evaluate the contribution of rare genetic variants to 414 plasma proteins. The frequency distribution of genetic variants is skewed towards the rare spectrum, and damaging variants are more often rare. We estimate that less than 4.3% of the narrow-sense heritability is expected to be explained by rare variants in our cohort. Using a gene-based approach, we identify Cis-associations for 237 of the proteins, which is slightly more compared to a GWAS (N = 213), and we identify 34 associated loci in Trans. Several associations are driven by rare variants, which have larger effects, on average. We therefore conclude that rare variants could be of importance for precision medicine applications, but have a more limited contribution to the missing heritability of complex diseases.

Abstract PD14-05: Portraying tumor evolution of lobular breast cancer through phylogenetic analysis

Abstract Background: Despite therapeutic advancements, a substantial number of patients with treated early-stage invasive lobular carcinoma (ILC) of the breast will still relapse as late as 20 years after the initial tumor diagnosis, thus making follow-up a challenging task. Here, using patient-matched primary and metastatic tumors from patients with ILC, we aimed to decipher the evolution of lobular breast cancer through time and explore metastatic dissemination events and underlying heterogeneity. Materials and Methods: We retrospectively identified 38 patients with metastatic ILC, of which 6 with de novo metastatic disease. In total, 99 formalin-fixed paraffin embedded tissue samples of primary tumor and metastasis, together with well-annotated clinicopathological data, were available. Low coverage whole-genome sequencing was performed to infer somatic copy number aberrations (CNA). Phylogenetic reconstruction was performed with Integer Linear Program for the Copy-Number Tree Problem algorithm. Results: Reconstruction of the phylogenetic trees showed different patterns of dissemination. For patients with multiple metastatic samples sequenced (4/4 patients), the metastases were found to be seeded from a common metastatic precursor. For a subset of patients with multiple primaries (5/10 patients), distant metastases were seeded from specific clones inside the primary tumor. Detailed analysis of known driver genes showed that alterations in common driver genes such as TP53, MYC and CCND1 were shared between primary tumor and metastases in a large proportion of the patients (47%, 32% and 21% of patients respectively) whilst CNA in other driver genes including FGFR2, PTEN or AKT2 among others, were frequently private in the metastases. Interestingly, we observed that in approximately 30% of the patients, the metastases harbored less CNA than the matched primary tumor, resulting in shorter metastatic branches and possibly indicating a decelerated tumor progression. Further analysis of the genomic and clinicopathological characteristics showed that these patients were mostly de novo metastatic (p-value=0.002), metastasizing primarily to the liver or bone and had significantly more aberrations in NF1, AKT1 and FGFR (p-value=0.04, 0.01 and 0.03 respectively), when compared to the rest of the cohort. Finally, most of the patients (63%) had a lower number of CNA accumulated after the metastatic lineage separation compared to the common trunk, implying a late dissemination event. Conclusions: In this study, we described different metastatic dissemination events taking place in ILC. Our analyses allowed us to identify a subset of tumors characterized by slow tumor progression, indicating potential tumor dormancy, as well as evidence of late dissemination evidence for most of the patients, thus highlighting the importance of early detection through cancer screening. These findings further shed light on how ILCs evolves through time and could potentially influence the clinical management of invasive lobular breast cancer. Citation Format: Danai Fimereli, David Venet, Mattia Rediti, David N Brown, Bram Boeckx, Marion Maetens, Samira Majjaj, Ghizlane Rouas, Maria Capra, Giuseppina Bonizzi, Federica Contaldo, Christine Galant, Martine Piccart, Giancarlo Pruneri, Denis Larsimont, Diether Lambrechts, Christine Desmedt, Françoise Rothé, Christos Sotiriou. Portraying tumor evolution of lobular breast cancer through phylogenetic analysis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-05.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

A rapidly reversible mutation generates subclonal genetic diversity and unstable drug resistance

Most genetic changes have negligible reversion rates. As most mutations that confer resistance to an adverse condition (e.g., drug treatment) also confer a growth defect in its absence, it is challenging for cells to genetically adapt to transient environmental changes. Here, we identify a set of rapidly reversible drug-resistance mutations in Schizosaccharomyces pombe that are caused by microhomology-mediated tandem duplication (MTD) and reversion back to the wild-type sequence. Using 10,000× coverage whole-genome sequencing, we identify nearly 6,000 subclonal MTDs in a single clonal population and determine, using machine learning, how MTD frequency is encoded in the genome. We find that sequences with the highest-predicted MTD rates tend to generate insertions that maintain the correct reading frame, suggesting that MTD formation has shaped the evolution of coding sequences. Our study reveals a common mechanism of reversible genetic variation that is beneficial for adaptation to environmental fluctuations and facilitates evolutionary divergence.

Skim sequencing: an advanced NGS technology for crop improvement

High-throughput genotyping has become more convenient and cost-effective due to recent advancements in next-generation sequencing (NGS) techniques. Numerous approaches exploring sequencing advances for genotyping have been developed over the past decade, which includes different variants of genotyping-by-sequencing (GBS), and restriction-site associated DNA sequencing (RAD-seq). Most of these methods are based on the reduced representation of the genome, which ultimately reduces the cost of sequencing by many folds. However, continuously lowering the cost of sequencing makes it more convenient to use whole genome-based approaches. In this regard, skim sequencing, where low coverage whole-genome sequencing is used for the identification of large numbers of polymorphic markers cost-effectively. In the present review, we have discussed recent technological advancements, applicability, and challenges of skim sequencing-based genotypic approaches for crop improvement programmes. Skim sequencing is being extensively used for genotyping in diverse plant species and has a wide range of applications, particularly in quantitative trait loci (QTL) mapping, genomewide association studies (GWAS), fine genetic map construction, and identification of recombination and gene conversion events in various breeding programmes. The cost-effectiveness, simplicity, and genomewide coverage will increase the application of skims sequencing-based genotyping. The article summarizes the protocol, uses, bioinformatics tools, its application, and future prospects of skim sequencing in crop improvement.

XGAP: a python based efficient, modular, extensible and fault tolerant genomic analysis pipeline for variant discovery.

Since the first human genome was sequenced in 2001, there has been a rapid growth in the number of bioinformatic methods to process and analyze next-generation sequencing (NGS) data for research and clinical studies that aim to identify genetic variants influencing diseases and traits. To achieve this goal, one first needs to call genetic variants from NGS data, which requires multiple computationally intensive analysis steps. Unfortunately, there is a lack of an open-source pipeline that can perform all these steps on NGS data in a manner, which is fully automated, efficient, rapid, scalable, modular, user-friendly and fault tolerant. To address this, we introduce xGAP, an extensible Genome Analysis Pipeline, which implements modified GATK best practice to analyze DNA-seq data with the aforementioned functionalities. xGAP implements massive parallelization of the modified GATK best practice pipeline by splitting a genome into many smaller regions with efficient load-balancing to achieve high scalability. It can process 30× coverage whole-genome sequencing (WGS) data in ∼90min. In terms of accuracy of discovered variants, xGAP achieves average F1 scores of 99.37% for single nucleotide variants and 99.20% for insertion/deletions across seven benchmark WGS datasets. We achieve highly consistent results across multiple on-premises (SGE & SLURM) high-performance clusters. Compared to the Churchill pipeline, with similar parallelization, xGAP is 20% faster when analyzing 50× coverage WGS on Amazon Web Service. Finally, xGAP is user-friendly and fault tolerant where it can automatically re-initiate failed processes to minimize required user intervention. xGAP is available at https://github.com/Adigorla/xgap. Supplementary data are available at Bioinformatics online.