Gene conversion explains elevated diversity in the immunity modulating APL1 gene of the malaria vector Anopheles funestus

The leucine rich repeat gene APL1 is a key component of immunity to Plasmodium and other microbial pathogens in Anopheles mosquitoes. In the malaria vector Anopheles funestus the APL1 gene has four paralogues which occur along the same chromosome arm. We show that APL1 has exceptional levels of non-synonymous polymorphism across the range of An. funestus with an average πn of 0.027 versus a genome-wide average of 0.002, and πn (and πs) is consistently high in populations across Africa. The pattern of APL1 diversity was consistent between independent pooled-template and target-enrichment datasets, however no link between APL1 diversity and insecticide-resistance was observed with the phenotyped target-enrichment dataset. Two further innate immunity genes of the gambicin anti-microbial peptide family had πn/πs ratios greater than one, possibly driven by either positive or balancing selection. Cecropin antimicrobial peptides were expressed much more highly than other anti-microbial peptide genes, an observation discordant with current models of anti-microbial peptide activity. The observed APL1 diversity likely results from gene conversion between paralogs, as evidenced by shared polymorphisms, overlapping read mappings, and recombination events among paralogues. Gene conversion at APL1 versus alternative explanations is concordant with similarly elevated diversity in APL1 and TEP1 loci in An. gambiae. In contrast, the more closely related An. stephensi which also encodes a single-copy of APL1 does not show this elevated diversity. We hypothesise that a more open chromatin formation at the APL1 locus due to higher gene expression than its paralogues enhances gene conversion, and therefore increased polymorphism, at APL1.

SNP and Phylogenetic Characterization of Low Viral Load SARS-CoV-2 Specimens by Target Enrichment

Background: Surveillance of SARS-CoV-2 across the globe has enabled detection of new variants and informed the public health response. With highly sensitive methods like qPCR widely adopted for diagnosis, the ability to sequence and characterize specimens with low titers needs to keep pace.Methods: Nucleic acids extracted from nasopharyngeal swabs collected from four sites in the United States in early 2020 were converted to NGS libraries to sequence SARS-CoV-2 genomes using metagenomic and xGen target enrichment approaches. Single nucleotide polymorphism (SNP) analysis and phylogeny were used to determine clade assignments and geographic origins of strains.Results: SARS-CoV-2-specific xGen enrichment enabled full genome coverage for 87 specimens with Ct values <29, corresponding to viral loads of >10,000 cp/ml. For samples with viral loads between 103 and 106 cp/ml, the median genome coverage for xGen was 99.1%, sequence depth was 605X, and the “on-target” rate was 57 ± 21%, compared to 13%, 2X and 0.001 ± 0.016%, respectively, for metagenomic sequencing alone. Phylogenetic analysis revealed the presence of most clades that existed at the time of the study, though clade GH dominated in the Midwest.Conclusions: Even as vaccines are being widely distributed, a high case load of SARS-CoV-2 infection persists around the world. Viral genetic surveillance has succeeded in warning the public of new variants in circulation and ensured that diagnostic tools remain resilient to a steadily increasing number of mutations. Target capture offers a means of characterizing low viral load samples which would normally pose a challenge for metagenomic sequencing.

hybpiper-rbgv and yang-and-smith-rbgv: Containerization and additional options for assembly and paralog detection in target enrichment data

PREMISE: The HybPiper pipeline has become one of the most widely used tools for the assembly of target enrichment (sequence capture) data for phylogenomic analysis. Between the production of locus sequences and phylogenetic analysis, the identification of paralogs is a critical step ensuring accurate inference of evolutionary relationships. Algorithmic approaches using gene tree topologies for the inference of ortholog groups are computationally efficient and broadly applicable to non-model organisms, especially in the absence of a known species tree. Unfortunately, software compatibility issues, unfamiliarity with relevant programming languages, and the complexity involved in running numerous subsequent analysis steps continue to limit the broad uptake of these approaches and constrain their application in practice. METHODS AND RESULTS: We updated the scripts constituting HybPiper and a pipeline for the inference of ortholog groups ("Yang and Smith") to provide novel options for the treatment of supercontigs, remove bugs, and seamlessly use the outputs of the former as inputs for the latter. The pipelines were containerised using Singularity and implemented via two Nextflow pipelines for easier deployment and to vastly reduce the number of commands required for their use. We tested the pipelines with several datasets, one of which is presented for demonstration. CONCLUSIONS: hybpiper-rbgv and yang-and-smith-rbgv provide easy installation, user-friendly experience, and robust results to the phylogenetic community. They are presently used as the analysis pipeline of the Australian Angiosperm Tree of Life project. The pipelines are available at https://github.com/chrisjackson-pellicle.

Diagnostic Yield and Therapeutic Consequences of Targeted Next-Generation Sequencing in Sporadic Primary Immunodeficiency

<b><i>Introduction:</i></b> Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders characterized by increased susceptibility to infections, immune dysregulation, and/or malignancy. Genetic studies, especially during the last decade, led to a better understanding of the pathogenesis of primary immunodeficiencies and contributed to their classification into distinct monogenic disorders falling under one of the &#x3e;430 currently known inborn errors of immunity (IEI). The growing availability of molecular genetic testing resulted in the increasing identification of patients with IEI. Here, we evaluated the diagnostic yield and the clinical consequences of targeted next-generation sequencing (tNGS) in a cohort of 294 primary immunodeficiency patients, primarily consisting of cases with sporadic primary antibody deficiency. <b><i>Method:</i></b> We have custom designed a tNGS panel to sequence a cohort of PID patients. Agilent’s HaloPlex Target Enrichment System for Illumina was used for DNA target enrichment. <b><i>Results:</i></b> tNGS identified a definite or predicted pathogenic variant in 15.3% of patients. The highest diagnostic rate was observed among patients with combined immunodeficiency or immune dysregulation, for whom genetic diagnosis may affect therapeutic decision-making. <b><i>Conclusion:</i></b> Next-generation sequencing has changed diagnostic assignment and paved the way for targeted therapeutic intervention with agents directed at reverting the disease-causing molecular abnormality or its pathophysiological consequences. Therefore, such targeted therapies and identifying the genetic basis of PID can be essential for patients with manifested immune dysregulation as conventional immunomodulatory regimens may exert an immunosuppressive effect, aggravating their immunodeficiency or may only inadequately control autoimmune or lymphoproliferative manifestations.

Exploring the Prognostic Value and Biological Function of CPNE1 Gene in Hepatocellular Carcinoma

BackgroundLiver hepatocellular carcinoma (LIHC), the major histology subtype of primary liver cancer, accounts for 70-80% proportion of total liver cancer cases. Copine1 (CPNE1), the first discovered CPNE1 family member, participates in the process of carcinogenesis and development of diverse tumors. Our study aimed to investigate the expression and prognostic value of CPNE1 gene in hepatocellular carcinoma (HCC), to explore its functional network in HCC and its effects on biological behaviors such as proliferation, migration and invasion of HCC cells, and to explore its related signaling pathways.METHODSHCCDB, CCLE and HPA online databases were used to explore the expression of CPNE1 gene in HCC tissues; LinkedOmics online database was used to analyze the co-expression network of CPNE1 in hepatocellular carcinoma, and gene set enrichment analysis (GSEA) was used for GO functional annotation, KEGG pathway enrichment analysis, kinase target enrichment, miRNA target enrichment and transcription factor target enrichment analysis. The expression levels of CPNE1 in normal hepatocytes and several hepatocellular carcinoma cell lines were detected by RT-qPCR, and finally HepG2 and MHCC-97H cells were selected to construct CPNE1 knockdown cell lines by transfection with siRNA, and the knockdown efficiency was detected by Western Blot and RT-qPCR. The effect of CPNE1 knockdown on the proliferation of hepatocellular carcinoma cells was examined by CCK8 assay and clone formation assay; the effect of CPNE1 knockdown on the migration ability of hepatocellular carcinoma cells was assessed by cell scratch assay and Transwell cell migration assay; finally, the expression of related signaling pathway proteins was examined by Western Blot. The correlation of CPNE1 expression with immune infiltration and immune checkpoint molecules in HCC tissues was analyzed using TIMER online database.RESULTSAnalysis in several databases showed that CPNE1 was highly expressed in HCC tissues and significantly correlated with sex, age, cancer stage and tumor grade. Overall survival (OS) was significantly lower in patients with high CPNE1 expression than in patients with low CPNE1 expression, and CPNE1 could be used as an independent prognostic indicator for HCC. GSEA analysis showed that co-expressed genes of CPNE1 were mainly involved in biological processes such as establishment of protein localization to membrane, ribonucleoprotein complex biogenesis and lipid localization. Q-PCR showed that CPNE1 expression was upregulated in HCC cells compared with normal hepatocytes, and knockdown of CPNE1 gene inhibited the AKT/P53 pathway, resulting in decreased proliferation, migration and invasion of HCC cells. The level of CPNE1 expression in HCC was significantly and positively correlated with the level of infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells (p<0.001), and with the expression of immune checkpoint molecules PDCD1, CD274, CTLA4, LAG3, HAVCR2, and TIGIT.CONCLUSIONThe expression of CPNE1 was significantly higher in HCC tissues than in normal liver tissues, and high CPNE1 expression was associated with poor prognosis. Knockdown of CPNE1 inhibited AKT/P53 pathway activation and suppressed HCC cell proliferation and migration. There was a significant correlation between CPNE1 expression and tumor immune infiltration in HCC.

Using herbarium samples for NGS methods – a methodological comparison

Herbaria harbor a tremendous amount of plant specimens that are rarely used for plant systematic studies. The main reason is the difficulty to extract a decent quantity of good quality DNA from the preserved plant material. While the extraction of ancient DNA in animals is well established, studies including old plant material are still underrepresented. In our study we compared the standard Qiagen DNeasy Plant Mini Kit and a specific PTB-DTT protocol on to two different plant genera (Xanthium L. and Salix L.). The included herbarium material covered about two centuries of plant collections. A selected subset of samples was used for a standard library preparation as well as a target enrichment approach. The results revealed that PTB-PTT resulted in higher quantity and quality regarding DNA yield. Despite the lower overall yield of DNA, the Qiagen Kit resulted in better sequencing results regarding the number of filtered and mapped reads. We were able to successfully sequence a sample from 1820 and conclude that it is possible to include old herbarium specimens in NGS approaches. This opens a treasure box in phylogenomic research.

Target Enrichment Enhances the Sensitivity of Sanger Sequencing for BRAF V600 Mutation Detection

BRAF is a serine/threonine protein kinase whose mutations lead to unregulated cell growth and cause different types of cancers. Since V600E is a major BRAF mutation and V600E detection as a companion diagnostic test (CDx) is stipulated in the labeling of the BRAF V600 inhibitors. Traditional Sanger sequencing cannot accurately detect mutations lower than 15% variant allele frequency (VAF) due to its limited sensitivity. Here we applied our patented XNA molecular clamping technology to modify Sanger sequencing template preparation by enriching the mutation population. We found that the use of our mutation-enriched template enhanced the analytical sensitivity of Sanger sequencing to 0.04% VAF. The method is verified to detect V600E, V600K, and V600R mutants and is validated for the known BRAF mutation status in clinical samples. Our streamlined protocol can be used for easy validation of the highly sensitive target-enrichment method for detecting BRAF V600 mutations using Sanger sequencing in clinical labs. In addition to BRAF V600 mutations, this method can be extended to the detection of other clinically important actionable mutations for cancer diagnostics.

Detection of Human Papillomavirus Integration in Brain Metastases from Oropharyngeal Tumors by Targeted Sequencing

Human papillomavirus (HPV) positive and negative head and neck squamous cell carcinoma (HNSCC) are known to have differential phenotypes, including the incidence and location of metastases. HPV positive (HPV+) HNSCC are more likely to metastasize to distant sites, such as the lung, brain, and skin. Among these locations, metastasis to the brain is a rare event, and little is known about specific risk factors for this phenotype. In this report, we describe two patients who developed brain metastases from HNSCC. Both patient tumors had p16INK4a overexpression, suggesting these tumors were HPV+. This was confirmed after PCR, in situ hybridization, and mass spectrometry detected the presence of HPV type 16 (HPV16) DNA, RNA and protein. To further characterize the presence of HPV16, we used a target enrichment strategy on tumor DNA and RNA to isolate the viral sequences from the brain metastases. Analysis by targeted next generation sequencing revealed that both tumors had the HPV genome integrated into the host genome at known hotspots, 8q24.21 and 14q24.1. Applying a similar target enrichment strategy to a larger cohort of HPV+ HNSCC brain metastases could help to identify biomarkers that can predict metastasis and/or identify novel therapeutic options.