Differences In Copy Number Research Articles

Modeling gene expression on the X chromosome in the brain for transcriptome‐wide association studies

AbstractBackgroundAnalysis of sex chromosomes is under‐reported in Alzheimer’s disease (AD) studies. As we gain more knowledge of disease associations and tissue‐specific regulatory effects of autosomal loci, there is a growing interest in building prediction models of gene expression in the brain to model the cumulative functions of non‐coding variants on the sex chromosomes. Transcriptome‐wide Association Studies (TWAS) can identify disease‐associated genes using these prediction models built for specific tissues by integrating genotype, imputed gene expression, and phenotype information. A key challenge of this approach for sex chromosome analysis is how to model copy number differences between males and females: genetic variants on the X chromosome are hemizygous for males, while females are heterozygous or homozygous diploid.MethodWe explore modeling approaches on the X chromosome using WGS and RNA‐seq data in 13 Brain sub‐regions retrieved from the GTEx project, currently restricting analyses to males. We used elastic‐net regression, with a balanced LASSO‐ridge penalty (mixing parameter α=0.5), as recommended by Wheeler et al. 2016. We tuned the penalization parameter λ for each gene in each tissue and assessed the model performance. There are 2392 genes on chromosome X, with only approximately 800 genes expressed in each GTEx brain tissue. SNPs (MAF>0.05) within the gene’s 1M bp flanking window were used to train the model. We used mean squared error to determine the penalization parameter and select the best models.ResultWe built 761 prediction models in 13 Brain sub‐regions on the expression of 503 genes. For example, we fit the prediction model of Gene COL4A6 (ENSG00000197565) in the Cortex, using 89 males. 83 cis‐SNPs were kept in the final model (λ=0.015). The model R2 is 0.20, suggesting that 20% of the variance of expression levels in males can be explained.ConclusionThese models predict the cis‐regulated gene expression on the X chromosome in males. We can then use these estimated gene expression weights to impute male expression levels to identify (putative) causal genes for AD with TWAS.

Aquatic insects differentially affect lake sturgeon larval phenotypes and egg surface microbial communities.

Documentation of how interactions among members of different stream communities [e.g., microbial communities and aquatic insect taxa exhibiting different feeding strategies (FS)] collectively influence the growth, survival, and recruitment of stream fishes is limited. Considerable spatial overlap exists between early life stages of stream fishes, including species of conservation concern like lake sturgeon (Acipenser fulvescens), and aquatic insects and microbial taxa that abundantly occupy substrates on which spawning occurs. Habitat overlap suggests that species interactions across trophic levels may be common, but outcomes of these interactions are poorly understood. We conducted an experiment where lake sturgeon eggs were fertilized and incubated in the presence of individuals from one of four aquatic insect FS taxa including predators, facultative and obligate-scrapers, collector-filterers/facultative predators, and a control (no insects). We quantified and compared the effects of different insect taxa on the taxonomic composition and relative abundance of egg surface bacterial and lower eukaryotic communities, egg size, incubation time to hatch, free embryo body size (total length) at hatch, yolk-sac area, (a measure of resource utilization), and percent survival to hatch. Mean egg size varied significantly among insect treatments. Eggs exposed to predators had a lower mean percent survival to hatch. Eggs exposed to predators had significantly shorter incubation periods. At hatch, free embryos exposed to predators had significantly smaller yolk sacs and total length. Multivariate analyses revealed that egg bacterial and lower eukaryotic surface community composition varied significantly among insect treatments and between time periods (1 vs 4 days post-fertilization). Quantitative PCR documented significant differences in bacterial 16S copy number, and thus abundance on egg surfaces varied across insect treatments. Results indicate that lethal and non-lethal effects associated with interactions between lake sturgeon eggs and free embryos and aquatic insects, particularly predators, contributed to lake sturgeon trait variability that may affect population levels of recruitment.

DNA read count calibration for single-molecule, long-read sequencing

There are many applications in which quantitative information about DNA mixtures with different molecular lengths is important. Gene therapy vectors are much longer than can be sequenced individually via short-read NGS. However, vector preparations may contain smaller DNAs that behave differently during sequencing. We have used two library preparations each for Pacific Biosystems (PacBio) and Oxford Nanopore Technologies NGS to determine their suitability for quantitative assessment of varying sized DNAs. Equimolar length standards were generated from E. coli genomic DNA. Both PacBio library preparations provided a consistent length dependence though with a complex pattern. This method is sufficiently sensitive that differences in genomic copy number between DNA from E. coli grown in exponential and stationary phase conditions could be detected. The transposase-based Oxford Nanopore library preparation provided a predictable length dependence, but the random sequence starts caused the loss of original length information. The ligation-based approach retained length information but read frequency was more variable. Modeling of E. coli versus lambda read frequency via cubic spline smoothing showed that the shorter genome could be used as a suitable internal spike-in for DNAs in the 200 bp to 10 kb range, allowing meaningful QC to be carried out with AAV preparations.

THE NATURE OF EMBRYONIC MOSAICISM ACROSS AGE AND CLINICS: AN ANALYSIS OF 21,345 PGT-A CYCLES

Preimplantation genetic testing for aneuploidy (PGT-A) was developed to prevent transferring embryos with chromosomal abnormalities. However, current genetic testing is sensitive enough to detect mosaic abnormalities, or non-uniform chromosomal copy number differences within a subset of trophectoderm cells. An absolute threshold for clinically significant mosaicism remains poorly defined and clinics differ in their management of mosaic embryo results. Here we share our efforts to uncover clinically relevant features of mosaicism via in depth analysis of a multinational database.

Role of N-acetylcysteine treatment in women with advanced age undergoing IVF/ICSI cycles: A prospective study.

ObjectiveThe main objective of this study was to explore the efficacy of a new antioxidant N-acetylcysteine (NAC) supplementation in reproductive outcomes of advanced age women undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET), and the effect on the expression of L-glutathione (GSH) in follicular fluid (FF) and mitochondrial DNA (mtDNA) copy number of granulosa cells.MethodsThe present prospective randomized controlled study was conducted in 200 patients with advanced age women undergoing GnRH antagonist protocol. The treatment group (group A) consisted of 100 women who received N-acetylcysteine treatment from the menstrual phase of the previous cycle for about 45 days using the GnRH antagonist protocol. The control group (group B) consisted of 100 women who received the same protocol without N-acetylcysteine. Total gonadotrophin dosage the number of oocyte received, high-quality blastocysts, and pregnancy outcomes were compared between two groups. Pregnancy outcomes included biochemical pregnancy rate, clinical pregnancy rate, embryo implantation rate, ectopic pregnancy rate, multiple pregnancy rate, and ongoing pregnancy rate. Follicular fluid (FF) was collected after oocytes were gathered. The GSH content in the FF was tested with enzyme linked immunosorbent assay (ELISA). The mtDNA copy number of the granulosa cells was measured using real-time PCR techniques.ResultsTotal doses of Gn in the NAC treatment group were less than those in the control group (2385.50 ± 879.19 vs. 2527.63 ± 1170.33, P = 0.047). Compared with the control, the number of high-quality blastocysts in NAC treatment increased significantly (1.82 ± 2.12 vs. 1.43 ± 1.58, p = 0.014). Clinical pregnancy rates did not differ in both groups (all P > 0.05). At the same time, the GSH content in the FF differed significantly between the two groups (1.88 ± 1.23 vs. 1.07 ± 0.70, p = 0.001). There was no significant difference in the mtDNA copy number between the two groups (P = 0.157).ConclusionA combination of NAC and Gn treatment is capable of improving the ovarian response to superovulation drugs in assisted reproductive technologies (ARTs) and also in aged populations. The addition of NAC during IVF can improve the quality of blastocysts in advanced age female subjects. However, more clinical trials are required to be designed to confirm this conclusion in future.Ethics and disseminationThe experiment solicited approval from the Institutional ethics committee of the Affiliated Reproductive Hospital of Shandong University. All the participants provided written informed consent. This survey was conducted as per the Declaration of Helsinki and relevant amendments.Trial registration numberwww.chictr.org.cn, identifier ChiCTR2100048297.

From bioinformatic screening of genetic markers to low invasive lymph node metastases diagnosis in patients with cervical cancer

Background. The problem of lymph nodes metastases diagnosing in cervical cancer remains relevant and not fully resolved. The last decade studies results have shown the great potential of molecular markers in lymph nodes metastasis prediction, however, additional studies for their implementation in clinical practice are required.
 Aim. Bioinformatic and laboratory screening of molecular markers of cervical tumors regional metastasis for its low invasive diagnosis.
 Material and methods. The study was performed on 400 patients with cervical cancer and 40 donors without oncological pathology. To identify potential molecular markers of lymph node metastatic lesions, the Cancer Genome Atlas database was initially analyzed. The identified markers were validated by the Real-Time-polymerase chain reaction in tumor cell samples (extracted using laser microdissection) and extracellular deoxyribonucleic acid (DNA). The MannWhitney test was used to assess the differences; the Bonferroni correction was used to account for multiple comparisons.
 Results. At the bioinformation stage, the change in the copy number of 5493 genes was analyzed, of which 79 genes were selected that most often change their copy number. During the data validation, it was found that primary tumor cells and tumor cells of metastases from the lymph nodes differ from normal cervix cells in the level of gene copies. The copy number of the CCND1 and PPARGC1A genes has the highest potential for regional lymph nodes metastases diagnosing in patients with cervical cancer; the PIK3CA, SPEN, ERBB3, APC, MUC4, CASP8, HLA-A, IGSF1 and TMTC1 loci have a lower potential. The EP300, TTN, DMD, DST, LAMP3, TORC2, TP53 and FOXO3 genes can be used to diagnose cervical cancer, whether metastatic or not. Additional validation of markers was carried out on extracellular DNA of blood plasma of cervical cancer patients and conditionally healthy donors. The presence of a differential copy number of PIK3CA, SPEN, ERBB3, APC, CCND1, HLA-A, TTN, MUC4, DST, PPARGC1A genes was found in two groups of patients with cervical cancer with and without metastatic lesions of the lymph nodes.
 Conclusion. The study made it possible to form a list of potential molecular markers for low invasive diagnostics of cervical cancer in general (EP300, LAMP3, TORC2, FOXO3, TP53) and cervical cancer with metastatic lesions of regional lymph nodes (PIK3CA/DST, APC/PPARGC1A, ERBB3/HLA-A and LAMP3/MUC4).

Comparative analysis of mitochondrial genomes of maize CMS-S subtypes provides new insights into male sterility stability

BackgroundCytoplasmic male sterility (CMS) is a trait of economic importance in the production of hybrid seeds. In CMS-S maize, exerted anthers appear frequently in florets of field-grown female populations where only complete male-sterile plants were expected. It has been reported that these reversions are associated with the loss of sterility-conferring regions or other rearrangements in the mitochondrial genome. However, the relationship between mitochondrial function and sterility stability is largely unknown.ResultsIn this study, we determined the ratio of plants carrying exerted anthers in the population of two CMS-S subtypes. The subtype with a high ratio of exerted anthers was designated as CMS-Sa, and the other with low ratio was designated as CMS-Sb. Through next-generation sequencing, we assembled and compared mitochondrial genomes of two CMS-S subtypes. Phylogenetic analyses revealed strong similarities between the two mitochondrial genomes. The sterility-associated regions, S plasmids, and terminal inverted repeats (TIRs) were intact in both genomes. The two subtypes maintained high transcript levels of the sterility gene orf355 in anther tissue. Most of the functional genes/proteins were identical at the nucleotide sequence and amino acid sequence levels in the two subtypes, except for NADH dehydrogenase subunit 1 (nad1). In the mitochondrial genome of CMS-Sb, a 3.3-kilobase sequence containing nad1-exon1 was absent from the second copy of the 17-kb repeat region. Consequently, we detected two copies of nad1-exon1 in CMS-Sa, but only one copy in CMS-Sb. During pollen development, nad1 transcription and mitochondrial biogenesis were induced in anthers of CMS-Sa, but not in those of CMS-Sb. We suggest that the impaired mitochondrial function in the anthers of CMS-Sb is associated with its more stable sterility.ConclusionsComprehensive analyses revealed diversity in terms of the copy number of the mitochondrial gene nad1-exon1 between two subtypes of CMS-S maize. This difference in copy number affected the transcript levels of nad1 and mitochondrial biogenesis in anther tissue, and affected the reversion rate of CMS-S maize. The results of this study suggest the involvement of mitochondrial robustness in modulation of sterility stability in CMS-S maize.

Hybrid incompatibility between Drosophila virilis and D. lummei is stronger in the presence of transposable elements.

Mismatches between parental genomes in selfish elements are frequently hypothesized to underlie hybrid dysfunction and drive speciation. However, because the genetic basis of most hybrid incompatibilities is unknown, testing the contribution of selfish elements to reproductive isolation is difficult. Here, we evaluated the role of transposable elements (TEs) in hybrid incompatibilities between Drosophila virilis and D. lummei by experimentally comparing hybrid incompatibility in a cross where active TEs are present in D. virilis (TE+) and absent in D. lummei, to a cross where these TEs are absent from both D. virilis (TE-) and D. lummei genotypes. Using genomic data, we confirmed copy number differences in TEs between the D. virilis (TE+) strain and both the D. virilis (TE-) strain and D. lummei. We observed F1 postzygotic reproductive isolation exclusively in the interspecific cross involving TE+ D. virilis but not in crosses involving TE- D. virilis. This mirrors intraspecies dysgenesis where atrophied testes only occur when TE+ D. virilis is the paternal parent. A series of backcross experiments, that accounted for alternative models of hybrid incompatibility, showed that both F1 hybrid incompatibility and intrastrain dysgenesis are consistent with the action of TEs rather than genic interactions. Thus, our data suggest that this TE mechanism manifests as two different incompatibility phenotypes. A further Y-autosome interaction contributes to additional, sex-specific, inviability in one direction of this cross-combination. These experiments demonstrate that TEs that cause intraspecies dysgenesis can increase reproductive isolation between closely related lineages, thereby adding to the processes that consolidate speciation.

The improved genome of the nematode Parapristionchus giblindavisi provides insights into lineage-specific gene family evolution.

Nematodes such as Caenorhabditis elegans and Pristionchus pacificus are extremely successful model organisms for comparative biology. Several studies have shown that phenotypic novelty but also conserved processes are controlled by taxon-restricted genes. To trace back the evolution of such new or rapidly evolving genes, a robust phylogenomic framework is indispensable. Here, we present an improved version of the genome of Parapristionchus giblindavisi which is the only known member of the sister group of Pristionchus. Relative to the previous short-read assembly, the new genome is based on long reads and displays higher levels of contiguity, completeness, and correctness. Specifically, the number of contigs dropped from over 7,303 to 735 resulting in an N50 increase from 112 to 791 kb. We made use of the new genome to revisit the evolution of multiple gene families. This revealed Pristionchus-specific expansions of several environmentally responsive gene families and a Pristionchus-specific loss of the de novo purine biosynthesis pathway. Focusing on the evolution of sulfatases and sulfotransferases, which control the mouth form plasticity in P. pacificus, reveals differences in copy number and genomic configurations between the genera Pristionchus and Parapristionchus. Altogether, this demonstrates the utility of the P. giblindavisi genome to date and polarizes lineage-specific patterns.

DiNAMIC.Duo: detecting somatic DNA copy number differences without a normal reference.

MotivationSomatic DNA copy number alterations (CNAs) arise in tumor tissue because of underlying genomic instability. Recurrent CNAs that occur in the same genomic region across multiple independent samples are of interest to researchers because they may contain genes that contribute to the cancer phenotype. However, differences in copy number states between cancers are also commonly of interest, for example when comparing tumors with distinct morphologies in the same anatomic location. Current methodologies are limited by their inability to perform direct comparisons of CNAs between tumor cohorts, and thus they cannot formally assess the statistical significance of observed copy number differences or identify regions of the genome where these differences occur.ResultsWe introduce the DiNAMIC.Duo R package that can be used to identify recurrent CNAs in a single cohort or recurrent copy number differences between two cohorts, including when neither cohort is copy neutral. The package utilizes Python scripts for computational efficiency and provides functionality for producing figures and summary output files.Availability and implementationThe DiNAMIC.Duo R package is available from CRAN at https://cran.r-project.org/web/packages/DiNAMIC.Duo/index.html. This article uses publicly available data from the Broad Institute TCGA Genome Data Analysis Center, https://doi.org/10.7908/C11G0KM9.Supplementary information Supplementary data are available at Bioinformatics online.

Insertion sequences and other mobile elements associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteraemia.

Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in the dissemination and modification of the antibiotic-resistant phenotype. To that end, we leveraged fully resolved Enterococcus faecium and Enterococcus faecalis genomes of isolates collected over 5 days from an inpatient with prolonged bacteraemia. Isolates from both species harboured similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events and IS-mediated excision events in plasmids of E. faecium isolates. We also characterize a novel arrangement of the ISs in a Tn1546-like transposon in E. faecalis isolates likely implicated in a vancomycin genotype–phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in liaSR genes and a putative composite transposon in E. faecium , offering a new paradigm for the study of daptomycin resistance and novel insights into its dissemination. In conclusion, our study highlights the role ISs and other transposable elements play in the rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.

Organellar transcripts dominate the cellular mRNA pool across plants of varying ploidy levels

Mitochondrial and plastid functions depend on coordinated expression of proteins encoded by genomic compartments that have radical differences in copy number of organellar and nuclear genomes. In polyploids, doubling of the nuclear genome may add challenges to maintaining balanced expression of proteins involved in cytonuclear interactions. Here, we use ribo-depleted RNA sequencing (RNA-seq) to analyze transcript abundance for nuclear and organellar genomes in leaf tissue from four different polyploid angiosperms and their close diploid relatives. We find that even though plastid genomes contain <1% of the number of genes in the nuclear genome, they generate the majority (69.9 to 82.3%) of messenger RNA (mRNA) transcripts in the cell. Mitochondrial genes are responsible for a much smaller percentage (1.3 to 3.7%) of the leaf mRNA pool but still produce much higher transcript abundances per gene compared to nuclear genome. Nuclear genes encoding proteins that functionally interact with mitochondrial or plastid gene products exhibit mRNA expression levels that are consistently more than 10-fold lower than their organellar counterparts, indicating an extreme cytonuclear imbalance at the RNA level despite the predominance of equimolar interactions at the protein level. Nevertheless, interacting nuclear and organellar genes show strongly correlated transcript abundances across functional categories, suggesting that the observed mRNA stoichiometric imbalance does not preclude coordination of cytonuclear expression. Finally, we show that nuclear genome doubling does not alter the cytonuclear expression ratios observed in diploid relatives in consistent or systematic ways, indicating that successful polyploid plants are able to compensate for cytonuclear perturbations associated with nuclear genome doubling.

A new method for determining ribosomal DNA copy number shows differences between Saccharomyces cerevisiae populations

Ribosomal DNA genes (rDNA) encode the major ribosomal RNAs and in eukaryotes typically form tandem repeat arrays. Species have characteristic rDNA copy numbers, but there is substantial intra-species variation in copy number that results from frequent rDNA recombination. Copy number differences can have phenotypic consequences, however difficulties in quantifying copy number mean we lack a comprehensive understanding of how copy number evolves and the consequences. Here we present a genomic sequence read approach to estimate rDNA copy number based on modal coverage to help overcome limitations with existing mean coverage-based approaches. We validated our method using Saccharomyces cerevisiae strains with known rDNA copy numbers. Application of our pipeline to a global sample of S. cerevisiae isolates showed that different populations have different rDNA copy numbers. Our results demonstrate the utility of the modal coverage method, and highlight the high level of rDNA copy number variation within and between populations.

Changes to the mtDNA copy number during yeast culture growth.

We show that the mitochondrial DNA (mtDNA) copy number in growing cultures of the yeast Saccharomyces cerevisiae increases by a factor of up to 4, being lowest (approx. 10 per haploid genome) and stable during rapid fermentative growth, and highest at the end of the respiratory phase. When yeast are grown on glucose, the onset of the mtDNA copy number increase coincides with the early stages of the diauxic shift, and the increase continues through respiration. A lesser yet still substantial copy number increase occurs when yeast are grown on a nonfermentable carbon source, i.e. when there is no diauxic shift. The mtDNA copy number increase during and for some time after the diauxic shift is not driven by an increase in cell size. The copy number increase occurs in both haploid and diploid strains but is markedly attenuated in a diploid wild isolate that is a ready sporulator. Strain-to-strain differences in mtDNA copy number are least apparent in fermentation and most apparent in late respiration or stationary phase. While changes in mitochondrial morphology and function were previously known to accompany changes in physiological state, it had not been previously shown that the mtDNA copy number changes substantially over time in a clonal growing culture. The mtDNA copy number in yeast is therefore a highly dynamic phenotype.

Duck hepatitis A virus type 1 mediates cell cycle arrest in the S phase

BackgroundDuck hepatitis A virus type 1 (DHAV-1) is one of the most serious pathogens endangering the duck industry. However, there are few studies on the regulation of the cell cycle by DHAV-1.MethodsIn this study, flow cytometry was applied to analyze the effect of DHAV-1 infection on the cell cycle of duck embryo fibroblasts (DEFs). Subsequently, we analyzed the effects of cell cycle phases on DHAV-1 replication by real-time reverse transcriptase quantitative PCR (real-time RT-qPCR).ResultsFlow cytometry data analysis found that DEFs in the S phase increased by 25.85% and 54.21% at 24 h and 48 h after DHAV-1 infection, respectively. The levels of viral RNA detected by real-time RT-qPCR were higher in the DEFs with synchronization in the S phase or G0/G1 phase than in the control group. However, there was no difference in viral copy number between the G2/M phase arrest and control groups. In addition, non-structural protein 3D of DHAV-1 significantly increased cells in the S phase, indicating that 3D protein is one of the reasons for the cell cycle arrest in the S phase.ConclusionsIn summary, DHAV-1 infection induces the cell cycle arrest of DEFs in the S phase. Both S phase and G0/G1 phase synchronization facilitate the replication of DHAV-1, and 3D protein is one of the reasons for the S phase arrest.

Propidium monoazide-quantitative PCR to detect viable Legionella spp. in the supply process of tap water

Abstract Legionella are common in the aquatic environment and are responsible for legionellosis including severe pneumonia and Pontiac fever. The culture method has some limitations in quickly detecting viable Legionella. Therefore, we optimized real-time PCR (qPCR) combined with propidium monoazide (PMA) to quantify viable Legionella in the supply process of tap water, considering factors such as PMA concentration, length of the target gene, and turbidity of water samples. Among 30, 50, 100, and 200 μM PMA concentration, 100 μM PMA had the greatest difference in copy number between PMA-treated live and dead cells while minimizing the cytotoxic effect on live cells. Among the various sizes of the target gene (108, 386, 456, and 654 bp), the primer in 386 bp size effectively excluded dead cells without loss of qPCR efficiency. As a result of applying the PMA-qPCR method to samples including river, purified water, and tap water, live and dead cells could be distinguished for samples with turbidity of less than 10 NTU. The optimized PMA-qPCR can be a useful method of rapidly detecting viable Legionella spp. in the process of supplying tap water, and contributing to tap water that is safe from pathogens.

Abstract 6217: TP53 loss with whole genome doubling mediates heterogeneous intra-patient therapy response in EGFR-driven lung adenocarcinoma: A TRACERx study

Abstract Introduction: Mutations in the epidermal growth factor receptor (EGFR) are often found in never-smokers who develop lung adenocarcinoma (LUAD) and resistance to receptor-targeted tyrosine kinase inhibitors (TKIs) generally occurs within five years of treatment. Mixed responses, where individual tumor lesions within the same patient respond differently to treatment, contribute to early treatment failure and indicate the involvement of multiple genomic alterations. TP53 loss of function has been associated with both tolerance to chromosomal instability (CIN) and with shortened progression free survival in EGFR-driven tumors. The aim of this study was to investigate the hypothesis that CIN, together with loss of p53 function, may lead to diverse genotypes that underlie the mixed responses observed in EGFR-driven LUAD. Experimental Procedures: We used genetically engineered mouse models (GEMMs), driven by EGFR with or without concomitant Trp53 loss and human isogenic cell lines to investigate cellular evolution and the effect of whole genome doubling on targeted therapy responses and mechanisms of resistance. Next generation sequencing and shallow whole genome single cell analyses, together with longitudinal imaging analysis from the Aura clinical trials (AURA2, AURA3 and the AURA extension cohort, Identifiers: NCT02094261, NCT02151981 and NCT01802632) was used to investigate the effect of p53 loss on tumor evolution. Results: EGFR mutant tumors with clonal Trp53 loss or TP53 pathway disruption displayed increased Weighted Genome Integrity Index (wGII) and higher cell to cell variation in both the mouse and TRACERx data sets. We found that TP53 loss of function increased the incidence of mixed responses and resistance to targeted therapy in both mouse and human tumors leading to early treatment failure and reduced survival. Whole-exome sequencing (median depth of 92x, range: 58-169x) of nine erlotinib-resistant EGFR mouse tumors identified four EGFR bypassing mutations (oncogenic KRAS mutations; Q61H, Q61R, and two G12D mutations) and one likely driver mutation in FGFR2 (C286R). We could only identify one known resistance associated mutation, EGFRT790M, in EGFR mutant tumors with concomitant loss of Trp53. In depth analysis revealed no major copy number differences in treatment naÏve vs resistant EGFR mutant tumors. In contrast, 70% of all tumors with concomitant Trp53 loss had amplified a region of mouse chromosome 7, harboring MET and BRAF. Investigating an isogenic EGFR/TP53 mutant cell model system revealed whole genome doubling as advantageous in overcoming the selection pressure induced by targeted therapy. Conclusion: We find that loss of TP53 in the context of mutated EGFR leads to an altered and plastic genomic landscape, with multiple copy number changes, which in turn facilitates therapy resistance. Citation Format: Sebastijan Hobor, Maise Al Bakir, Marcin Skrzypski, Alexander M. Frankell, Bjorn Bakker, Thomas B. Watkins, Aleksandra Markovets, Jonathan R. Dry, Andrew P. Brown, Jasper van der Aart, Dahmane Oukrif, Marco Novelli, Matthew J. Renshaw, William Hill, Hilda van den Bos, Diana C. Spierings, Juliann Chmielecki, Carl Barrett, Kevin Litchfield, Elza de Bruin, Floris Foijer, Karen H. Vousden, TRACERx consortium, Robert E. Hynds, Crispin T. Hiley, Nnennaya Kanu, Simone Zaccaria, Eva C. Gronroos, Charles Swanton. TP53 loss with whole genome doubling mediates heterogeneous intra-patient therapy response in EGFR-driven lung adenocarcinoma: A TRACERx study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6217.

Quantification of antibiotic resistance genes in Siberian sturgeons (Acipenser baerii) fed Hermetia illucens-based diet

Siberian sturgeon (Acipenser baerii) represents a high-value fish farmed for obtaining high-priced food. In this study, the occurrence of antibiotic resistance (AR) genes encoding for resistance to antimicrobials administered in clinical practice, as aminoglycosides, β-lactams, macrolide-lincosamide-streptogramin B, tetracyclines, and vancomycin, were studied in the gut of sturgeons fed a diet containing 50% Hermetia illucens larvae by qPCR assays. Erm(A), erm(B), erm(C), tet(K), tet(M), tet(S), and tet(W) were detected in all samples. Feed and guts of sturgeons at beginning of rearing were negative for tet(O), vanA, vanB, mecA, and aac-aph. BlaZ was exclusively detected in control diet. The highest copy numbers were observed for tet(M), tet(K) and tet(S) in control diet. The copy numbers of most of the genes were higher in control diet than in diet containing 50% insect. Despite the differences in the gene copy numbers in control or insect-based diet, the results for AR genes contained in the guts of fish highlighted that, at 50% substitution level, no effect of the insect-based diet on the AR genes seems to occur in the experimental sturgeons at the end of rearing.

Response of Soil Fungal Communities in Diversified Rotations of Wheat and Different Crops

Crop-soil microorganism interactions and feedback are critical to soil health and crop production. The aim of this study was to clarify the difference in soil fungal communities under diversified rotations of wheat and different crops in the North China Plain and to provide a theoretical basis for the construction and optimization of ecological sustainable planting systems. The soil fungal community abundance, composition, and diversity of continuous winter wheat-summer maize M, winter wheat-summer peanut (summer maize) PM, and winter wheat-summer soybean (summer maize) SM treatments were studied using real-time quantitative PCR and high-throughput sequencing technology. The results showed that, compared with those of the continuous winter wheat-summer maize treatment, the peanut rotation treatment PM2 and soybean rotation treatment SM2 significantly reduced soil fungal ITS sequence copy numbers (P<0.05); there was no significant difference in soil fungal ITS sequence copy numbers between other rotation treatments and those of the control (P>0.05). Rotation treatments with peanut or soybean increased soil fungal community richness (Chao1 and ACE indices) and diversity (Shannon and InvSimson indices), in which the community richness of all rotation treatments and the community diversity of SM1/SM2 treatments varied significantly (P<0.05). The result of non-metric multidimensional scaling (NMDS) analysis showed that the soil fungal community among different rotation crops were obviously separated. The rotation crops significantly affected soil fungal community structure (PERMANOVA:r2=0.350, P=0.001; ANOSIM:r=0.478, P=0.001). Ascomycota (73.67%-85.48%) was the dominant phylum, whereas Sordariomycetes (30.53%-48.19%) and Eurotiomycetes (11.12%-31.19%) were the dominant classes of the fungal communities of sandy-loam fluvo-aquic soil in the North China Plain. There were significantly different taxa of soil fungal communities in different rotations. Potential pathogens such as Neocosmospora, Plectosphaerella, and Gibellulopsis were significantly enriched in the rotations of winter wheat-summer peanut (summer maize), whereas potential beneficial fungi such as Penicillium and Zopfiella were significantly enriched in the rotations of winter wheat-summer soybean (summer maize). Compared with that under the continuous winter wheat-summer maize treatment, rotations with peanut or soybean increased the relative abundance of pathotroph, pathotroph-symbiotroph, and saprotroph-symbiotroph fungi and decreased the relative abundance of saprotroph fungi. The soil fungal community richness and structure were significantly related to soil organic carbon and available nutrients, and the Shannon diversity index was significantly related to soil mineral nitrogen and available phosphorus. In summary, on the basis of continuous winter wheat-summer maize rotation in the North China Plain, adding summer peanut or summer soybean instead of summer maize for rotations with different interval years could increase the richness and diversity of soil fungal communities and significantly change soil fungal community structure. In particular, summer soybean as the preceding crop had a positive effect on the enrichment of potential beneficial fungi.

Circulating KRAS variant-specific shedding and association with survival in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) receiving chemoimmunotherapy.

2548 Background: Circulating tumor DNA (ctDNA) is increasingly used as a prognostic marker with high ctDNA shedding associated with poor survival. Gene-, but not variant-specific, differences in ctDNA shedding have been reported. Tumor burden, mitotic rate, and cell death rate have been proposed as contributors to ctDNA shedding. Here we investigate associations of ctDNA shedding for the two most common mPDAC KRAS variants, G12D and G12V, with tumor burden, mitotic score, and overall survival (OS). Methods: Pretreatment (baseline) ctDNA was analyzed by droplet digital PCR for 86 (including 44 G12D, 30 G12V) patients with mPDAC receiving front-line chemoimmunotherapy in a randomized open-label Phase II study (NCT03214250). Baseline tumor burden in total, within the pancreas, and distally, was assessed by sum of RECIST target lesion diameters. Tumor tissue variant allele fraction (tVAF) and mitotic score (geometric mean expression of 65 mitosis-associated genes) were calculated from DNA and RNA sequencing. Results: ctKRAS shedding (dichotomized at median mutant copies/mL) was associated with OS for G12D bearing tumors (p = 0.03) but not G12V (p = 0.17, log-rank test). To identify variant-specific features of shedding, we examined the Spearman correlation for total tumor burden and ctKRAS shedding; G12D but not G12V shedding was correlated with tumor burden (p = 0.01 and p = 0.22 respectively). However, the higher tVAF in G12V compared to G12D tumors (p = 0.048, Mann-Whitney test) could result from differences in purity, ploidy, and KRAS copy number. Thus, we used tVAF as a scalar to calculate an adjusted tumor burden which was significantly correlated with both G12D and G12V ctDNA shedding (p = 0.004 and 0.02, respectively). When a patient’s distal vs. pancreatic lesions were analyzed separately, pancreatic tumor burden was not correlated with G12D or G12V shedding (p = 0.10 and 0.33, respectively) but distal burden was correlated with both (p = 0.001 and 0.02, respectively). While there was no difference by KRAS variant for the correlation between adjusted tumor burden and shedding, these results do suggest that, in patients with mPDAC, distal rather than primary tumor burden may drive ctDNA shedding. Finally, tumor mitotic rate was combined with adjusted total tumor burden in a linear regression model; both were significant for predicting G12D shedding (p = 0.007 and p &lt; 0.0001, respectively) but not for G12V (p = 0.045 and p = 0.16, respectively). Conclusions: These data suggest that ctDNA shedding and survival associations may be KRAS variant-specific in mPDAC. Tumor mitotic score and location of tumors may explain some variant-specific differences in shedding. As clinical ctDNA tests become more widely used, further investigation of variant-specific shedding in KRAS and other genes may be key for proper interpretation of ctDNA tests.