Single Nucleotide Variant Analysis Research Articles

Identification of three small nucleolar RNAs (snoRNAs) as potential prognostic markers in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a non-Hodgkin lymphoma with high mortality rates. Small nucleolar RNAs (snoRNAs) are tumor-specific biological markers, but there are few studies on the role of snoRNAs in DLBCL. Survival-related snoRNAs were selected to construct a specific snoRNA-based signature via computational analyses (Cox regression and independent prognostic analyses) to predict the prognosis of DLBCL patients. To assist in clinical applications, a nomogram was built by combining the risk model and other independent prognostic factors. Pathway analysis, gene ontology analysis, transcription factor enrichment, protein-protein interactions, and single nucleotide variant analysis were used to explore the potential biological mechanisms of co-expressed genes. Twelve prognosis-correlated snoRNAs were selected from the DLBCL patient cohort of microarray profiles, and a three-snoRNA signature consisting of SNORD1A, SNORA60, and SNORA66 was constructed. DLBCL patients could be divided into high-risk and low-risk cohorts using the risk model, and the high-risk group and activated B cell-like (ABC) type DLBCL were linked with disappointing survival. In addition, SNORD1A co-expressed genes were inseparably linked to the biological functions of the ribosome and mitochondria. Potential transcriptional regulatory networks have also been identified. MYC and RPL10A were the most mutated SNORD1A co-expressed genes in DLBCL. Put together, our findings explored the potential biological effects of snoRNAs in DLBCL, and provided a new predictor for DLBCL prediction.

A pancancer analysis of CDK12 alterations in Chinese population.

e15068 Background: Cyclin-dependent kinases ( CDKs) are key regulators of both cell cycle progression and transcription. Analysis of mutation detection of CDK12 in pan-cancer could contribute to understand the complexity and diversity of genetic alterations present in CDK12 mutant cancers. Herein, we provide a pan-cancer analysis of CDK12 alterations in Chinese population. Methods: Using gene-panel target-capture next generation sequencing, we analyzed 17509 tumor tissue or plasma ctDNA samples from different patients. Testing included analysis of single nucleotide variants, insertions/deletions, fusions and amplifications. CDK12 variants of uncertain significance and synonymous variants excluded. Results: In total, 0.95% (167/17509) of pan-cancer patients harbored CDK12 mutation, and 32 patients carried at least two CDK12 mutations. The most common genetic alteration type was truncating mutations (128, 64%), followed by amplications (53, 26%), missense mutations (9, 5%) and splicing mutations (9, 5%). In our cohort, the highest CDK12 mutation frequency tumor type was prostate cancer(11.30%), followed by breast cancer (3.96%), bladder cacner (3.16%), stomach cancer (1.84%) and colorectal cacner (1.13%). The TCGA pan-cancer cohort confirmed that the CDK12-altered cancer was associated with shorter overall survival (81.11 vs 133.00 mos, p = 5.30×10−18) in prostate cancer. Tumor mutation burdens(TMB) were calculated for 137 samples of all the 167 samples. The median TMB was 7.66 mut/Mb with 38%≥10 mut/Mb and 17%≥20 mut/Mb. including a patient with hypermutated in colorectal cancer(TMB: 88.24 Mut/Mb), harboring three CDK12 mutations. Conclusions: Genomic alterations in CDK12 are widespread among Chinese patients, with alteration rates and alteration types varying by cancer type. We identified prostate cancer as the cancer type with the highest prevalence of CDK12 oncogenic alterations. Additional analyses are ongoing.

Persistence of an epidemic cluster of Rhodotorula mucilaginosa in multiple geographic regions in China and the emergence of a 5-flucytosine resistant clone

ABSTRACT Rhodotorula mucilaginosa, an environmental yeast widely used in industry and agriculture, is also an opportunistic pathogen resistant to multi-antifungals. During the national surveillance in China, R. mucilaginosa has been documented from various hospitals and regions. At present, the molecular epidemiology of invasive infections caused by R. mucilaginosa and their resistance profiles to antifungals were unknown. Here we collected 49 strains from four hospitals located in different geographic regions from 2009 to 2019 in China, determined their genotypes using different molecular markers and quantified susceptibilities to various antifungals. Sequencing of ITS and D1/D2 regions in rDNA indicated that 73.5% (36/49) of clinical strains belong to same sequence type (rDNA type 2). Microsatellite (MT) genotyping with 15 (recently developed) tandem repeat loci identified 5 epidemic MT types, which accounted for 44.9% (22/49) of clinical strains, as well as 27 sporadic MT types. Microsatellite data indicated that the presence of an epidemic cluster including 35 strains (71.4%) repeatedly isolated in four hospitals for eight years. Single nucleotide variants (SNVs) from the whole genome sequence data also supported the clustering of these epidemic strains due to low pairwise distance. In addition, phylogenetic analysis of SNVs from these clinical strains, together with environmental and animal strains showed that the closely related epidemic cluster strains may be opportunistic, zoonotic pathogens. Also, molecular data indicated a possible clonal transmission of pan echinocandins-azoles-5-flucytosine resistant R. mucilaginosa strains in hospital H01. Our study demonstrated that R. mucilaginosa is a multi-drug resistant pathogen with the ability to cause nosocomial infection.

BiTSC 2: Bayesian inference of tumor clonal tree by joint analysis of single-cell SNV and CNA data

The rapid development of single-cell DNA sequencing (scDNA-seq) technology has greatly enhanced the resolution of tumor cell profiling, providing an unprecedented perspective in characterizing intra-tumoral heterogeneity and understanding tumor progression and metastasis. However, prominent algorithms for constructing tumor phylogeny based on scDNA-seq data usually only take single nucleotide variations (SNVs) as markers, failing to consider the effect caused by copy number alterations (CNAs). Here, we propose BiTSCn}{}^2, Bayesian inference of Tumor clonal Tree by joint analysis of Single-Cell SNV and CNA data. BiTSCn}{}^2 takes raw reads from scDNA-seq as input, accounts for the overlapping of CNA and SNV, models allelic dropout rate, sequencing errors and missing rate, as well as assigns single cells into subclones. By applying Markov Chain Monte Carlo sampling, BiTSCn}{}^2 can simultaneously estimate the subclonal scCNA and scSNV genotype matrices, subclonal assignments and tumor subclonal evolutionary tree. In comparison with existing methods on synthetic and real tumor data, BiTSCn}{}^2 shows high accuracy in genotype recovery, subclonal assignment and tree reconstruction. BiTSCn}{}^2 also performs robustly in dealing with scDNA-seq data with low sequencing depth and variant missing rate. BiTSCn}{}^2 software is available at https://github.com/ucasdp/BiTSC2.

Site-specific insertion of endonuclease recognition sites into amplicons to improve post-PCR analysis sensitivity of gene mutation

Precise detection of low-frequency gene mutations surrounded by excess wild-type DNA is important in many aspects of medical fields. Most hybridization-based methods for high-resolution mutant allele analysis are hindered by competition of the complementary strand with single-strand probes for the target strand. Here, we demonstrate that site-specific insertion of endonuclease recognition sites into amplicons allows post-PCR generation of short dsDNA or ssDNA, whereby improves the sensitivity of both melting temperature analysis (MTA) and end-point detection following up. Using a three-staged PCR protocol, enrichment of target gene and incorporation of specific restriction sites in amplicons were ensued with hardly any loss in amplification efficiency and specificity. It enables simultaneous discrimination among a panel of totally 11 EGFR 19 exon deletion mutations via MTA after post-PCR digestion by either FokI only or cooperated with CRISPR-Cas12a, using SYBR green I. By replacement of one double-strand cleavage site with a nickase binding domain post-PCR generation of ssDNA of interest via strand displacement amplification (termed as iSDA) is realized. Our preliminary investigation shows that iSDA permits analysis of single nucleotide variants down to 0.1% allelic-frequency using end-point detection. Given the good compatibility with the majority of mutant-enrich PCR methods, we envision it would advance the current gene profiling technologies to a large extent.

Variant interpretation: UCSC Genome Browser Recommended Track Sets.

The UCSC Genome Browser has been an important tool for genomics and clinical genetics since the sequence of the human genome was first released in 2000. As it has grown in scope to display more types of data it has also grown more complicated. The data, which are dispersed at many locations worldwide, are collected into one view on the Browser, where the graphical interface presents the data in one location. This supports the expertise of the researcher to interpret variants in the genome. Because the analysis of single nucleotide variants and copy number variants require interpretation of data at very different genomic scales, different data resources are required. We present here several Recommended Track Sets designed to facilitate the interpretation of variants in the clinic, offering quick access to datasets relevant to the appropriate scale.

Virulence Genotype and Phenotype of Multiple Antimicrobial-Resistant Escherichia coli Isolates from Broilers Assessed from a “One-Health” Perspective

Extraintestinal pathogenic Escherichia coli (ExPEC) include several serotypes that have been associated with colibacillosis in poultry and with urinary tract infections (UTIs) and newborn meningitis in humans. In this study, 57 antimicrobial-resistant E. coli from apparently healthy broiler chickens were characterized for their health and safety risks. These isolates belonged to 12 serotypes, and isolates of the same serotype were clonal based on single nucleotide variant analysis. Most of the isolates harbored plasmids; IncC and IncFIA were frequently detected. The majority of the resistant isolates harbored plasmid-mediated resistance genes, including aph(3″)-Ib, aph(6)-Id, blaCMY-2, floR, sul1, sul2, tet(A), and tet(B), in agreement with their resistant phenotypes. The class 1 integron was detected in all E. coli serotypes except O124:H25 and O7:H6. Of the 57 broiler E. coli isolates, 27 were avian pathogenic, among which 18 were also uropathogenic E. coli and the remainder were other ExPEC. The two isolates of serotype O161:H4 (ST117) were genetically related to the control avian pathogenic strains and a clinical isolate associated with UTIs. A strain of serotype O159:H45 (ST101) also was closely related to a UTI isolate. The detected virulence factors included adhesins, invasins, siderophores, type III secretion systems, and toxins in combination with other virulence determinants. A broiler isolate of serotype O7:H18 (ST38) carried the ibeA gene encoding a protein involved in invasion of brain endothelium on a 102-kbp genetic island. This isolate moderately adhered and invaded Caco-2 cells and induced mortality (42.5%) in a day-old-chick infection model. The results of this study suggest that multiple antimicrobial-resistant E. coli isolates recovered from apparent healthy broilers can be pathogenic and act as reservoirs for antimicrobial resistance genes, highlighting the necessity of their assessment in a "One-Heath" context.

Circulating Tumor Cell Transcriptomics as Biopsy Surrogates in Metastatic Breast Cancer

BackgroundMetastatic breast cancer (MBC) and the circulating tumor cells (CTCs) leading to macrometastases are inherently different than primary breast cancer. We evaluated whether whole transcriptome RNA-Seq of CTCs isolated via an epitope-independent approach may serve as a surrogate for biopsies of macrometastases.MethodsWe performed RNA-Seq on fresh metastatic tumor biopsies, CTCs, and peripheral blood (PB) from 19 newly diagnosed MBC patients. CTCs were harvested using the ANGLE Parsortix microfluidics system to isolate cells based on size and deformability, independent of a priori knowledge of cell surface marker expression.ResultsGene expression separated CTCs, metastatic biopsies, and PB into distinct groups despite heterogeneity between patients and sample types. CTCs showed higher expression of immune oncology targets compared with corresponding metastases and PB. Predictive biomarker (n = 64) expression was highly concordant for CTCs and metastases. Repeat observation data post-treatment demonstrated changes in the activation of different biological pathways. Somatic single nucleotide variant analysis showed increasing mutational complexity over time.ConclusionWe demonstrate that RNA-Seq of CTCs could serve as a surrogate biomarker for breast cancer macrometastasis and yield clinically relevant insights into disease biology and clinically actionable targets.

Deleterious Germline Variants Are Present in Patients with Myelodysplastic Syndrome of All Ages Treated with Related Allogeneic Stem Cell Transplantation

▪Inherited myeloid malignancies are included as a provisional category in the current World Health Organization classification and within clinical testing guidelines of the National Comprehensive Cancer Network for myelodysplastic syndrome (MDS) and the European LeukemiaNet. The frequency of deleterious germline variants in MDS patients diagnosed at or younger than 40 years old ranges from 15-20%. To determine the frequency of germline predisposition in MDS patients of all ages, we accessed peripheral blood samples collected by the Center for International Blood and Marrow Transplant Research (CIBMTR) from all MDS patients who had undergone allogeneic hematopoietic stem cell transplantation (HSCT) using related donors (Table 1).We performed whole exome sequencing in 404 donor/recipient pairs augmented with spike-in probes covering non-coding regions known to contain inherited risk alleles. Single nucleotide variants (SNVs) and copy number variants (CNVs) were interpreted according to American College of Medical Genetics and Genomics (ACMG), Association for Molecular Pathology, and Clinical Genome Sequence Variant Interpretation Working Group guidelines. Germline status was confirmed by the presence of a variant in the MDS patient and related donor and for variants previously only seen as germline alleles with a variant allele frequency of 40-60%.We examined variants in 236 genes associated with hematopoietic malignancies, bone marrow failure syndromes, immunodeficiencies and congenital/acquired cytopenias. We identified pathogenic germline variants in 28 out of 404 MDS patients (7%): 23 with autosomal dominant and five with autosomal recessive inheritance. MDS patients with deleterious germline variants were found in all age deciles, from age 11 to 71 (Figure 2A), and were more likely to develop higher-grade MDS (43% vs. 25%, p=0.04). Deleterious variants in 5 bone marrow failure syndrome-related genes were found predominantly in younger people, whereas middle-aged and senior MDS patients had most often deleterious variants in DDX41 (n=4) and cancer predisposition genes (n= 16; Figure 2B). Seventy-one percent of MDS patients with deleterious autosomal dominant germline variants shared that variant with their related donor. However, there were no differences in HSCT outcomes for these individuals.The 7% overall frequency of deleterious germline variants is likely an underestimate, because we were constrained in calling germline variants as those detectable in both the MDS patient and related donor, since only peripheral blood samples were available from the CIBMTR. Thus, we expect that 50% of related donors would not carry the variant. Moreover, because some germline predisposition disorders lead to cytopenias or other clinical features, some relatives with those variants may have been excluded as HSCT donors. We also expect that some variants deemed variants of uncertain significance are pathogenic, but will require segregation or functional studies to upgrade them into deleterious categories. Finally, our HSCT outcome measurements are underpowered due to the small numbers of MDS patients with each disorder.Based on the ACMG's recommendation to test clinical germline predisposition when positive findings are anticipated at >5%, our data support comprehensive germline genetic testing for all MDS patients regardless of their age at diagnosis or family history. Testing should include analysis of both SNVs and CNVs in genes and non-coding regions known to contain cancer predisposition alleles. Our data also justify a larger study to evaluate clinical impact of pathogenic germline variants on transplant outcomes. Until we know the impact of deleterious germline variants on HSCT outcomes, we continue to recommend avoiding using HSCT donors with such variants whenever possible given the risk of poor graft function/failure and donor-derived leukemias in HSCT recipients. [Display omitted] DisclosuresScott: Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Sobecks: CareDX: Membership on an entity's Board of Directors or advisory committees. Saber: Govt. COI: Other.

Use of medical exome sequencing for identification of underlying genetic defects in NICU: Experience in a cohort of 2303 neonates in China.

Emerging evidence demonstrates the clinical utility of genomic applications in newborn intensive care unit (NICU) patients with strong indications of Mendelian etiology. However, such applications' diagnostic yield and utility remain unclear for NICU cohorts with minimal phenotype selection. In this study, focused medical exome sequencing was used as a first-tier, singleton-focused diagnostic tool for 2303 unrelated sick neonates. Integrated analysis of single nucleotide variants (SNVs), small insertions and deletions (Indels), and large copy number variants (CNVs) was performed. The diagnostic rate in this NICU cohort is 12.3% (284/2303), with 190 probands with molecular diagnoses made from SNV/Indel analyses (66.9%), 93 patients with diagnostic aneuploidy/CNVs findings (32.8%), and 1 patient with both SNV and CNV (0.4%). In addition, 54 (2.3%) of patients had a reportable incidental finding. Multiple organ involvements, craniofacial abnormalities, and dermatologic abnormalities were the strongest positive predictors for a molecular diagnosis. Among the 190 cases with SNV/Indel defects, direct impacts on medical management were observed in 46.8% of patients after the results were reported. In this study, we demonstrate that focused medical exome sequencing is a powerful first-line diagnostic tool for NICU patients. Significant number of diagnosed NICU patients can benefit from more focused medical management and long-term care.

High-throughput sequencing of SARS-CoV-2 in wastewater provides insights into circulating variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) likely emerged from a zoonotic spill-over event and has led to a global pandemic. The public health response has been predominantly informed by surveillance of symptomatic individuals and contact tracing, with quarantine, and other preventive measures have then been applied to mitigate further spread. Non-traditional methods of surveillance such as genomic epidemiology and wastewater-based epidemiology (WBE) have also been leveraged during this pandemic. Genomic epidemiology uses high-throughput sequencing of SARS-CoV-2 genomes to inform local and international transmission events, as well as the diversity of circulating variants. WBE uses wastewater to analyse community spread, as it is known that SARS-CoV-2 is shed through bodily excretions. Since both symptomatic and asymptomatic individuals contribute to wastewater inputs, we hypothesized that the resultant pooled sample of population-wide excreta can provide a more comprehensive picture of SARS-CoV-2 genomic diversity circulating in a community than clinical testing and sequencing alone. In this study, we analysed 91 wastewater samples from 11 states in the USA, where the majority of samples represent Maricopa County, Arizona (USA). With the objective of assessing the viral diversity at a population scale, we undertook a single-nucleotide variant (SNV) analysis on data from 52 samples with >90% SARS-CoV-2 genome coverage of sequence reads, and compared these SNVs with those detected in genomes sequenced from clinical patients. We identified 7973 SNVs, of which 548 were “novel” SNVs that had not yet been identified in the global clinical-derived data as of 17th June 2020 (the day after our last wastewater sampling date). However, between 17th of June 2020 and 20th November 2020, almost half of the novel SNVs have since been detected in clinical-derived data. Using the combination of SNVs present in each sample, we identified the more probable lineages present in that sample and compared them to lineages observed in North America prior to our sampling dates. The wastewater-derived SARS-CoV-2 sequence data indicates there were more lineages circulating across the sampled communities than represented in the clinical-derived data. Principal coordinate analyses identified patterns in population structure based on genetic variation within the sequenced samples, with clear trends associated with increased diversity likely due to a higher number of infected individuals relative to the sampling dates. We demonstrate that genetic correlation analysis combined with SNVs analysis using wastewater sampling can provide a comprehensive snapshot of the SARS-CoV-2 genetic population structure circulating within a community, which might not be observed if relying solely on clinical cases.

Whole genome sequencing in the diagnosis of primary ciliary dyskinesia

BackgroundIt is estimated that 1–13% of cases of bronchiectasis in adults globally are attributable to primary ciliary dyskinesia (PCD) but many adult patients with bronchiectasis have not been investigated for PCD. PCD is a disorder caused by mutations in genes required for motile cilium structure or function, resulting in impaired mucociliary clearance. Symptoms appear in infancy but diagnosis is often late or missed, often due to the lack of a “gold standard” diagnostic tool and non-specific symptoms. Mutations in > 50 genes account for around 70% of cases, with additional genes, and non-coding, synonymous, missense changes or structural variants (SVs) in known genes presumed to account for the missing heritability.MethodsUK patients with no identified genetic confirmation for the cause of their PCD or bronchiectasis were eligible for whole genome sequencing (WGS) in the Genomics England Ltd 100,000 Genomes Project. 21 PCD probands and 52 non-cystic fibrosis (CF) bronchiectasis probands were recruited in Wessex Genome Medicine Centre (GMC). We carried out analysis of single nucleotide variants (SNVs) and SVs in all families recruited in Wessex GMC.Results16/21 probands in the PCD cohort received confirmed (n = 9), probable (n = 4) or possible (n = 3) diagnosis from WGS, although 13/16 of these could have been picked up by current standard of care gene panel testing. In the other cases, SVs were identified which were missed by panel testing. We identified variants in novel PCD candidate genes (IFT140 and PLK4) in 2 probands in the PCD cohort. 3/52 probands in the non-CF bronchiectasis cohort received a confirmed (n = 2) or possible (n = 1) diagnosis of PCD. We identified variants in novel PCD candidate genes (CFAP53 and CEP164) in 2 further probands in the non-CF bronchiectasis cohort.ConclusionsGenetic testing is an important component of diagnosing PCD, especially in cases of atypical disease history. WGS is effective in cases where prior gene panel testing has found no variants or only heterozygous variants. In these cases it may detect SVs and is a powerful tool for novel gene discovery.

Skin dysbiosis in the microbiome in atopic dermatitis is site-specific and involves bacteria, fungus and virus

BackgroundMicrobial dysbiosis with increased Staphylococcus aureus (S. aureus) colonization on the skin is a hallmark of atopic dermatitis (AD), however most microbiome studies focus on bacteria in the flexures and the microbial composition at other body sites have not been studied systematically.ObjectivesThe aim of the study is to characterize the skin microbiome, including bacteria, fungi and virus, at different body sites in relation to AD, lesional state, and S. aureus colonization, and to test whether the nares could be a reservoir for S. aureus strain colonization.MethodsUsing shotgun metagenomics we characterized microbial compositions from 14 well defined skin sites from 10 patients with AD and 5 healthy controls.ResultsWe found clear differences in microbial composition between AD and controls at multiple skin sites, most pronounced on the flexures and neck. The flexures exhibited lower alpha-diversity and were colonized by S. aureus, accompanied by S. epidermidis in lesions. Malassezia species were absent on the neck in AD. Virus mostly constituted Propionibacterium and Staphylococcusphages, with increased abundance of Propionibacterium phages PHL041 and PHL092 and Staphylococcus epidermidis phages CNPH82 and PH15 in AD. In lesional samples, both the genus Staphylococcus and Staphylococcus phages were more abundant. S. aureus abundance was higher across all skin sites except from the feet. In samples where S. aureus was highly abundant, lower abundances of S. hominis and Cutibacterium acnes were observed. M. osloensis and M. luteus were more abundant in AD. By single nucleotide variant analysis of S. aureus we found strains to be subject specific. On skin sites some S. aureus strains were similar and some dissimilar to the ones in the nares.ConclusionsOur data indicate a global and site-specific dysbiosis in AD, involving both bacteria, fungus and virus. When defining targeted treatment clinicians should both consider the individual and skin site and future research into potential crosstalk between microbiota in AD yields high potential.

The coral symbiont Candidatus Aquarickettsia is variably abundant in threatened Caribbean acroporids and transmitted horizontally

The symbiont “Candidatus Aquarickettsia rohweri” infects a diversity of aquatic hosts. In the threatened Caribbean coral, Acropora cervicornis, Aquarickettsia proliferates in response to increased nutrient exposure, resulting in suppressed growth and increased disease susceptibility and mortality of coral. This study evaluated the extent, as well as the ecology and evolution of Aquarickettsia infecting threatened corals, Ac. cervicornis, and Ac. palmata and their hybrid (“Ac. prolifera”). Aquarickettsia was found in all acroporids, with coral host and geographic location impacting the infection magnitude. Phylogenomic and genome-wide single-nucleotide variant analysis of Aquarickettsia found phylogenetic clustering by geographic region, not by coral taxon. Analysis of Aquarickettsia fixation indices suggests multiple sequential infections of the same coral colony are unlikely. Furthermore, relative to other Rickettsiales species, Aquarickettsia is undergoing positive selection, with Florida populations experiencing greater positive selection relative to other Caribbean locations. This may be due in part to Aquarickettsia proliferating in response to greater nutrient stress in Florida, as indicated by greater in situ replication rates in these corals. Aquarickettsia was not found to significantly codiversify with either the coral animal or the coral’s algal symbiont (Symbiodinium “fitti”). Quantitative PCR analysis showed that gametes, larvae, recruits, and juveniles from susceptible, captive-reared coral genets were not infected with Aquarickettsia. Thus, horizontal transmission of Aquarickettsia via coral mucocytes or an unidentified host is more likely. The prevalence of Aquarickettsia in Ac. cervicornis and its high abundance in the Florida coral population suggests that coral disease mitigation efforts focus on preventing early infection via horizontal transmission.

Analysis of Single Nucleotide Variants (SNVs) Induced by Passages of Equine Influenza Virus H3N8 in Embryonated Chicken Eggs.

Vaccination is an effective method for the prevention of influenza virus infection. Many manufacturers use embryonated chicken eggs (ECE) for the propagation of vaccine strains. However, the adaptation of viral strains during subsequent passages can lead to additional virus evolution and lower effectiveness of the resulting vaccines. In our study, we analyzed the distribution of single nucleotide variants (SNVs) of equine influenza virus (EIV) during passaging in ECE. Viral RNA from passage 0 (nasal swabs), passage 2 and 5 was sequenced using next generation technology. In total, 50 SNVs with an occurrence frequency above 2% were observed, 29 of which resulted in amino acid changes. The highest variability was found in passage 2, with the most variable segment being IV encoding hemagglutinin (HA). Three variants, HA (W222G), PB2 (A377E) and PA (R531K), had clearly increased frequency with the subsequent passages, becoming dominant. None of the five nonsynonymous HA variants directly affected the major antigenic sites; however, S227P was previously reported to influence the antigenicity of EIV. Our results suggest that although host-specific adaptation was observed in low passages of EIV in ECE, it should not pose a significant risk to influenza vaccine efficacy.

Genomic instability-associated lncRNA signature predicts prognosis and distinct immune landscape in gastric cancer.

BackgroundCharacterized by multiple features, genomic stability-related markers, such as microsatellite instability (MSI), were regulated as an important predictor of chemotherapy and immunity responses in cancer treatment. The aim of our study was to identify a genomic instability-associated long non-coding RNA (lncRNA) signature to help predict the survival and therapy response of gastric cancers (GCs).MethodsWe used RNA sequencing and single nucleotide variant (SNV) data from The Cancer Genome Atlas-stomach adenocarcinoma (TCGA-STAD) datasets to explore genomic instability-associated lncRNAs. Hierarchical cluster analyses of 197 differentially expressed genomic instability-associated lncRNAs were performed to separate GC patients into two groups, namely, the genomically unstable (GU)-like group and the genomically stable (GS)-like group.ResultsCox regression analysis was conducted to finally identify six lncRNAs (LINC02678, HOXA10-AS, RHOXF1-AS1, AC010789.1, LINC01150, and TGFB2-AS1) with independent prognostic value to establish the genomic instability-associated lncRNA signature (GILncSig). Based on the SNV analysis, GILncSig was correlated with accumulation of gene mutation counts. Further comparisons between different risk score groups were performed to assess chemotherapy drug sensitivity and immune landscape variations.ConclusionsOur study not only revealed the genomic instability-associated lncRNAs in GCs, but provided a key method and resource for further studies of the role of these lncRNAs play, and introduced a potential new way to identify genomic instability-associated cancer biomarkers.

Unique transcriptome features of pea (<i>Pisum sativum</i> L.) lines with differing responses to beneficial soil microorganisms

BACKGROUND: Garden pea (Pisum sativum L.) possesses the ability to form beneficial symbioses with various soil microorganisms. However, different pea cultivars, genotypes, and lines gain more or less benefit from these interactions, so the trait named efficiency of interaction with soil microorganisms (EIBSM) was suggested to describe this phenomenon. The molecular mechanisms underlying the manifestation of the EIBSM trait are not properly studied, and only few works focusing on plant responses to combined microbial preparations have been published to date.
 METHODS: Eight pea lines previously described as contrasting in manifestation of the EIBSM trait were grown in pots with soil under combined inoculation with nodule bacteria and arbuscular mycorrhizal fungi, and the transcriptome profiles of the whole root systems of the plants were investigated using 3'MACE RNA sequencing.
 RESULTS: The relatedness of the lines inferred from the analysis of transcripts SNVs (Single Nucleotide Variants) corresponded to the manifestation of the EIBSM trait: three high-EIBSM lines and three low-EIBSM lines formed two distinct clusters. Thus, the gene expression profiles were compared between these two clusters, which enabled identification of transcriptome signatures characteristic for each group. The lines previously described as high-EIBSM have lower symbiotic activity, and the expression levels of pathogen response genes were elevated compared to the lines with low EIBSM.
 CONCLUSION: This result suggests that the mechanism of high interaction efficiency may be connected to stricter host control of symbionts, allowing such plants to expend less on the symbioses.

Reads in a haystack: extracting complete mitogenome sequences hidden in baculovirus datasets.

Thaumatotibia leucotreta (Lepidoptera, Tortricidae) is one of many economically important insect pests for which no complete mitogenome sequence is available. The complete mitochondrial sequences for this species and other key pests could assist in the development of novel molecular techniques, such as enabling the identification of population-specific markers which could assist in improved monitoring of populations. The objective of this study was to determine whether NGS datasets generated for entomopathogenic viruses contain reads originating from host mitochondrial DNA. A total of 28 NGS datasets generated for the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV) were analysed in this study. Three datasets contained sufficient reads providing adequate coverage for the assembly of complete mitogenomes. All 13 protein-coding genes, 22 tRNAs and both rRNAs present in the mitogenomes of other species within the Grapholitini tribe, were identified. Phylogenetic analysis of the mitogenomes at both an intrafamilial and interspecies level grouped the sequences within the Olethreutinae and T. leucotreta clades, respectively. Analysis of single nucleotide variations (SNVs) between each T. leucotreta sequence indicated up to 75 differences across the mitogenome. The methodology used in this study could be expanded to other baculovirus NGS datasets enabling the generation of novel lepidopteran mitogenome sequences.

Abstract 577: A unified pipeline to detect small mutations, structural variations and copy number alterations from targeted cell-free DNA sequencing in cancer

Abstract Introduction: Analysis of cell-free DNA (cfDNA) has recently emerged as a non-invasive modality for guiding cancer diagnostics and treatment decisions. However, efforts have predominantly focused on the analysis of single nucleotide variants (SNVs) and insertions/deletions (indels). Despite the clinical significance of many larger variants such as structural variations (SVs) and copy number alterations (CNAs), detecting them in cfDNA remains a challenge. Given the lack of existing tools we seek to develop an integrated bioinformatic pipeline for SV and CNA detection in cfDNA following targeted hybrid-capture next-generation sequencing (NGS), along with standard SNV and indel analysis. Methods: SVs were first detected using Manta, Lumpy and Delly in plasma cfDNA in comparison with matched peripheral blood leukocyte (PBL) DNA samples from cancer patients, then combined to identify consensus SVs and genotyped throughout samples from patients and healthy individuals. Next, consensus SVs were called somatic events if they were supported by split reads and discordant read pairs in cfDNA samples from patients but not in matched PBL or healthy donor cfDNA samples. For CNA analysis, the ratio of read depth between patient-derived plasma cfDNA and a panel of healthy controls was calculated across genomic bins using the CNVkit tool, followed by bias correction and recentralization using CNA negative control genes to account for read coverage imbalances in targeted NGS. Last, SNV and indel analysis was integrated from the CAPP-Seq pipeline. Results: We applied our pipeline to targeted hybrid-capture NGS data from 48 patients across two independent cohorts of metastatic castration resistant prostate cancer (mCRPC). The targeted panel covered the full-length AR gene body and a hotspot region of TMPRSS2-ERG fusion break points. Consistent with earlier whole genome studies, we confirmed known CNAs and SVs in tumor suppressors, oncogenes and regulatory elements including AR gene and AR enhancer duplications (22/48, 46% of patients), TMPRSS2-ERG gene fusions (9/48, 19%), PTEN and TP53 loss (8/48, 17%). Notably, our pipeline outperformed FACTERA which did not detect any TMPRSS2-ERG gene fusions or AR/enhancer tandem duplications. Subsequent analysis showed high concordance between plasma cfDNA and matched tumor biopsies, and our pipeline recapitulated the landscape of SVs and CNAs in an in silico cfDNA simulation from tumor biopsies. Finally, we showed that alterations of the AR/enhancer locus detected by our pipeline were strongly associated with treatment resistance, patient progression-free and overall survival in mCRPC. Conclusion: We developed a unified pipeline for detection of SVs, CNAs and small mutations in cfDNA targeted sequencing with potential applications in monitoring cancer progression and predicting patient treatment response. Citation Format: Ha X. Dang, Jace Webster, Pradeep S. Chauhan, Steven H. Hartman, Wenjia Feng, Elisa M. Ledet, Haley Ellis, Patrick J. Miller, Ellen B. Jaeger, Sydney A. Caputo, Peter K. Harris, A. Oliver Sartor, Russell K. Pachynski, Aadel A. Chaudhuri, Christopher A. Maher. A unified pipeline to detect small mutations, structural variations and copy number alterations from targeted cell-free DNA sequencing in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 577.

Interferon-γ Induces Distinct mRNA and Protein Profiles in Acute and Chronic Myeloid Leukemia

Leukemia cells promote tumor survival and progression by avoiding host immune surveillance. A key pathway involves indoleamine 2,3-dioxygenase-1 (IDO1), an interferon (IFN)-γ-inducible molecule mediating feedback inhibition and hindering anti-tumor immunity. We have previously shown thattreatment of primary acute myeloid leukemia (AML) cells with IFN-γ in vitro induces functional IDO1 in 50% of unselected cases (‘IFN-γ responders‘), an effect which was abrogated by STAT3 inhibitors. With a median follow-up of 8 years, these IFN-γ responders experienced a shorter event-free survival and overall survival. The current study was undertaken to dissect the intracellular signaling pathways that are activated in leukemia cells in response to IFN-γ.We employed an alpha version of the Vantage 3D™ DNA:RNA:Protein Heme Assay (NanoString Technologies Inc., Seattle, USA) to profile DNA mutations, RNA, protein and phosphoprotein simultaneously. The assay profiles 180 mRNA species involved in hematology/oncology pathways, 38 total and phosphorylated proteins (including JAK-STAT, Src, BTK, FLT3, PI3K-Akt, NF-kB), and 124 single nucleotide variants (SNVs) relevant to hematological malignancies (IDH, FLT3, KRAS, NRAS, DNMT3, JAK3 and others). K562 and TCC-S cells (chronic myeloid leukemia [CML] in blast crisis), KG1a cells (leukemia stem cell [LSC]-like AML) and Kasumi-1 cells [AML with t(8;21)] were stimulated with 100 ng/mL IFN-γ then harvested and lysed after 30m, 1h, 2h, 12h and 24h for processing in parallel workflows (protein/mRNA and DNA SNV assays) prior to overnight hybridization with NanoString reporters. Pooled RNA/DNA/protein samples were analyzed using the nCounter® FLEX platform and the nSolver® software (alpha version 4.0). CCRF-CEM and HUT78 cell lines, with known DNA variants and expression of protein targets represented in the 3D Heme panel, were used as controls.K562, TCC-S and Kasumi-1 cells, but not KG1a cells, up-regulated IDO1 mRNA after IFN-γ challenge. IFN-γ-sensitive CML cells (K562 and TCC-S) expressed high levels of mRNA transcripts for JAK-STAT intermediates and genes implicated in apoptosis, DNA damage and cytokine signaling (Fig. 1). IFN-γ-sensitive t(8;21) AML cells displayed a distinct transcriptomic profile with an up-regulated subset of IFN-inducible genes, including IRF1 , STAT1, BCL3, SOCS1 , PD-L1 , ISG15 , OSM , HLA class I and genes involved in antigen processing (CIITA ). In contrast, the transcriptome of IFN-γ-insensitive LSC-like KG1a cells consisted of high levels of genes implicated in epigenetics and transcription, NF-κB, MYC, B-cell receptor and T-cell receptor signaling, and low expression levels of genes mediating JAK-STAT signaling, apoptosis, DNA damage and cytokine signaling. Overall phosphorylation of intracellular proteins was largely unchanged after IFN-γ stimulation of KG1a cells. In contrast, IFN-γ activation of Kasumi-1 AML cells was associated with increased phosphorylation of Akt(S473), STAT3(Y705) and proline-rich Akt substrate of 40kD (PRAS40) (T246). Similar phosphorylation changes were apparent in TCC-S CML cells, although with lower magnitude. Interestingly, HOXA9 transcription factor, which is commonly dysregulated in acute leukemias, was highly expressed, both as mRNA and protein (r2=0.912; p<0.001), in LSC-like KG1a cells, but not in t(8;21) AML and CML cell lines. SNV analysis identified all expected gene mutations in the control cell lines and TP53(R248Q) in Kasumi-1 cells.The use of artificial neural network algorithms and a public collection of AML transcriptomic data (641 patients; E-MTAB-3444) allowed us to identify enriched functional-related gene groups (‘immune response’, ‘antigen processing and presentation’, ‘MAPK signaling pathway’; DAVID v6.8), the expression of which was predicted by the 3 most up-regulated transcripts in our IFN-γ-sensitive leukemia cells (IRF1, STAT1 and BCL3 ).In conclusion, the combined analysis of SNVs, mRNA and protein expression on the nCounter® platform is a powerful tool to characterize the molecular landscape of leukemias. Our analysis of IFN-γ-stimulated leukemia cells has identified molecular pathways that could be targeted in patients with ‘inflamed’ tumors, in which microenvironmental production of IFN-γ is heightened due to ongoing T-cell responses.Grant support: Roger Counter Foundation, UK; Qatar National Research Fund (#NPRP8-2297-3-494). [Display omitted] DisclosuresMeredith:NanoString Technologies: Employment. Ross:NanoString Technologies: Employment. Demirkan:NanoString Technologies: Employment. Rutella:NanoString Technologies, Seattle, USA: Research Funding.