Biology subjects

Hu, X.

Publications and source records attributed to Hu, X..

At least 325 records · Page 18Linked to original sources

bioRxiv2020

ScRNA-seq discover cell cluster change under OAB: ACE2 expression reveal possible alternation of 2019-nCoV infectious pathway

ObjectivePrevious study indicated that bladder cells which express ACE2 were a potential infection route of 2019-nCov. This study observed some differences of bladder cell cluster and their ACE2 expression between OAB mice and healthy mice, indicating the change of infectious possibility and pathway under overactive bladder (OAB) circumstance. Material and methodPubic dataset acquisition was used to get ACE2 expression in normal human bladder and mice bladder (GSE129845). We built up over OAB model and studied the impact on cell typing and ACE2 expression. By way of using single-cell RNA sequencing (scRNA-seq) technique, bladder cell clustering and ACE2 expression in various cell types were measured respectively. ResultIn pubic database (healthy human and mice bladder), ACE2 expression in humans and mice is concentrated in bladder epithelial cells. The disappearance of umbrella cells, a component of bladder epithelial, was found in our OAB model. In the two mouse bladder samples, ACE2 expression of epithelial cells is 34.1%, also the highest of all cell types. ConclusionThe disappearance of umbrella cell may alternate the infection pathway of 2019-nCov and relate to the onset and progression of OAB.

cell biology↗

bioRxiv2020

Donor-derived mitochondrial DNA variant peptides elicit allo-specific immune response in transplant patients

In stem cell transplant, mitochondrial DNA (mtDNA) nonsynonymous single nucleotide variants (SNVs) between donor and recipient (D-R) trigger alloimmune responses and transplant rejection. Whether similar alloimmune responses occurs in solid-organ transplantation remains unknown, particularly with the presence of human leukocyte antigen mismatches. This study characterized mtDNA SNVs between D-R of 163 human lung transplant pairs, and then, post-transplantation, assessed alloimmune responses against donor-derived mitochondrial peptides using ELISpot to measure interferon gamma (IFN{gamma}) release from recipients monocytes. We identified a median of 6 nonsynonymous mtDNA SNVs (Interquartile Range = 4 - 9) per D-R pair. SNVs were predominantly located at MT-CYB, MT-ATP6, and MT-ND3 genes. The number of SNVs was higher in D-R race non-concordant pairs than in race-concordant pairs. Donor-derived mitochondrial peptides triggered a 19.8-fold higher IFN{gamma} release compared to recipient-derived peptide. These findings were validated in heart transplantation and show that donor-derived mitochondrial peptides trigger allo-specific immune responses after transplantation.

genomics↗

bioRxiv2020

An OpenData portal to share COVID-19 drug repurposing data in real time

The National Center for Advancing Translational Sciences (NCATS) has developed an online open science platform - named the NCATS OpenData Portal (ODP) - for quickly and freely sharing complete NCATS translational datasets via an open-access, user-friendly interface. This paper describes the establishment of the ODP, initially deployed during the COVID-19 crisis, and provides a detailed analysis of COVID-19 drug repurposing screening datasets that served as the first large-scale use case for the platform. Over 10,000 compounds were tested across 17 quantitative high-throughput assays, covering a wide spectrum of the SARS-CoV-2 life cycle. In total, over 87,000 concentration-response curves and 426,000 data points were made publicly available on ODP in near real-time, enabling immediate access to complete datasets. The resource is flexible in accommodating various types and structures of data, and it has already expanded since its launch to host additional datasets for COVID-19, other viruses of pandemic potential, and beyond. The OpenData Portal has been designed as a scalable platform for real-time data sharing across drug discovery campaigns, regardless of disease area, with the overarching goal of accelerating discovery at NCATS, the NIH, and the greater scientific community.

microbiology↗

bioRxiv2020

An enormous Paris polyphylla genome sheds light on genome size evolution and polyphyllin biogenesis

The monocot family Melanthiaceae with varying genome sizes in a range of 230-fold is an ideal model to study the genome size fluctuation in plants. Its family member Paris genus demonstrates an evolutionary trend of bearing huge genomes characterized by an average c-value of 49.22 pg. Here, we report a 70.18 Gb genome assembly out of the 82.55 Gb genome of Paris polyphylla var. yunnanensis (PPY), which represents the biggest sequenced genome to date. We annotate 69.53% repetitive sequences in this genome and 62.50% of which are long-terminal repeat (LTR) transposable elements. Further evolution analysis indicates that the giant genome likely results from the joint effect of common and species-specific expansion of different LTR superfamilies, which might contribute to the environment adaptation after speciation. Moreover, we identify the candidate pathway genes for the biogenesis of polyphyllins, the PPY-specific medicinal saponins, by complementary approaches including genome mining, comprehensive analysis of 31 next-generation RNA-seq data and 55.23 Gb single-molecule circular consensus sequencing (CCS) RNA-seq reads, and correlation of the transcriptome and phytochemical data of five different tissues at four growth stages. This study not only provides significant insights into plant genome size evolution, but also paves the way for the following polyphyllin synthetic biology.

plant biology↗

bioRxiv2020

The SARS-CoV-2 cytopathic effect is blocked with autophagy modulators

SARS-CoV-2 is a new type of coronavirus capable of rapid transmission and causing severe clinical symptoms; much of which has unknown biological etiology. It has prompted researchers to rapidly mobilize their efforts towards identifying and developing anti-viral therapeutics and vaccines. Discovering and understanding the virus’ pathways of infection, host-protein interactions, and cytopathic effects will greatly aid in the design of new therapeutics to treat COVID-19. While it is known that chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including inhibiting autophagy, there are also dose-limiting toxicities in patients that make clearly establishing their potential mechanisms-of-action problematic. Therefore, we evaluated a range of other autophagy modulators to identify an alternative autophagy-based drug repurposing opportunity. In this work, we found that 6 of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero-E6 cells with EC50 values ranging from 2.0 to 13 µM and selectivity indices ranging from 1.5 to >10-fold. Immunofluorescence staining for LC3B and LysoTracker dye staining assays in several cell lines indicated their potency and efficacy for inhibiting autophagy correlated with the measurements in the SARS-CoV-2 cytopathic effect assay. Our data suggest that autophagy pathways could be targeted to combat SARS-CoV-2 infections and become an important component of drug combination therapies to improve the treatment outcomes for COVID-19.One Sentence Summary Blocking SARS-CoV-2 cytopathic effects with selective autophagy inhibitors underlying the clinical benefits of chloroquine and hydroxychloroquine.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

MAPK2 is a conserved Alveolate MAPK required for Toxoplasma cell cycle progression

Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The Apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, ERK7 and MAPKL1, have been respectively implicated in the regulation of conoid biogenesis and centrosome duplication. The third kinase, MAPK2, is specific to and conserved throughout Alveolata, though its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss-of-function in Toxoplasma. We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter-cell budding, which ultimately led to parasite death. MAPKL2-deficient parasites initiated, but did not complete DNA replication, and arrested prior to mitosis. Surprisingly, the parasites continued to grow in size and to replicate their Golgi, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest MAPK2 regulates a process at a distal site that is required for completion of centrosome duplication and initiation of parasite mitosis. ImportanceToxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, TgMAPK2 appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to Alveolata and not found in metazoa, and likely is a critical component of an essential parasite-specific signaling network.

cell biology↗

bioRxiv2020

HBV pgRNA can generate a circRNA with two junction sites

To verify whether Hepatitis B virus (HBV) can form circRNA, the circRNA sequencing results of the HBV-positive HepG 2.2.15 hepatocarcinoma cell line were compared with the HBV genome. A novel circRNA, named as HBV_circ_1, was mapped to the 489-2985 nt region of the HBV genome (GenBank accession No. KU668446.1), which was derived from HBV pgRNA and has two junction sites. A partial fragment of the pgRNA (corresponding to HBV DNA genome 489-3,182-2,985nt) was resected and then connected to form a junction site of HBV_circ_1 (corresponding to HBV DNA genome 489/2985nt). The 5-terminal region (corresponding to HBV DNA genome 1,820-1,932nt) of pgRNA was repeated with the region of pgRNA at the 3-terminal region. Another junction site of HBV_circ_1 (corresponding to HBV DNA genome 1820nt) was formed in a manner similar to DNA homologous recombination mediated by repeats of pgRNA. Moreover, this RNA homologous recombination mediated by the repeats of pgRNA does not have tissue specificity and genus specificity. Reverse transcription PCR, northern blotting, and tissue in situ hybridization confirmed the existence of HBV_circ_1 in HepG2.2.15 cells and HBV related hepatocellular carcinoma (HCC) tissue. This study expands our understanding of circRNA generation mechanisms, and provide a new perspective for understanding the molecular pathogenesis of the virus.

molecular biology↗

bioRxiv2020

Structure of SARS-CoV-2 main protease in the apo state reveals the inactive conformation

Mpro is of considerable interest as a drug target in the treatment of COVID-19 since the proteolytic activity of this viral protease is essential for viral replication. Here we report the first insight of the structure Mpro for SARS-CoV-2 in the inactive conformation under conditions close to the physiological state (pH 7.5) to an overall resolution of 1.9 [A]. The comparisons of Mpro in different states reveal that substrate binding site and the active site are more flexible in the inactive conformation than that in the active conformations. Notably, compared with the active conformation of the apo state structure in pH7.6 of SARS, the SARS-CoV-2 apo state is in the inactive conformation under condition close to physiological state (pH7.5). Two water molecules are present in the oxyanion hole in our apo state structure, whereas in the ligand-bound structure, water molecular is absence in the same region. This structure provides novel and important insights that have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational, structure-based, approaches for the design of specific SARS-CoV-2 ligands as new therapeutic agents.

biochemistry↗

bioRxiv2020

The conservation of a core virome in Aedes mosquitoes across different developmental stages and continents

Mosquitoes belonging to the genus Aedes can efficiently transmit many pathogenic arboviruses, placing a great burden on public health worldwide. In addition, they also carry a number of insect specific viruses (ISVs), and it was recently suggested that some of these ISVs might form a stable species-specific "core virome" in mosquito populations. However, little is known about such a core virome in laboratory colonies and if it is present across different developmental stages. In this study, we compared the viromes in eggs, larvae, pupae and adults of Aedes albopictus mosquitoes collected from the field as well as from a lab colony. The virome in lab-derived Ae. albopictus is very stable across all stages, consistent with a vertical transmission route of these viruses, forming a "vertically transmitted core virome". The different stages of field collected Ae. albopictus mosquitoes also contains this stable vertically transmitted core virome as well as another set of viruses shared by mosquitoes across different stages, which might be an "environment derived core virome". Both these vertically and environmentally transmitted core viromes in Ae. albopictus deserve more attention with respect to their effects on vector competence for important medically relevant arboviruses. To further study this core set of ISVs, we screened 46 publically available SRA viral metagenomic dataset of mosquitoes belonging to the genus Aedes. Some of the identified core ISVs are identified in the majority of SRAs. In addition, a novel virus, Aedes phasmavirus, is found to be distantly related to Yongsan bunyavirus 1, and the genomes of the core virus Phasi Charoen-like phasivirus is highly prevalent in the majority of the tested samples, with nucleotide identities ranging from 94% to 99%. Finally, Guadeloupe mosquito virus, and some related viruses formed three separated phylogenetic clades. How these core ISVs influence the biology of mosquito host, arboviruses infection and evolution deserve to be further explored.

microbiology↗

bioRxiv2020

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific protein targets in a proteasome-dependent manner. However, a major limitation to broader TPD applications is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent ligand for the E3 ligase RNF114. When linked to the BET family inhibitor JQ1, the resulting heterobifunctional PROTAC molecule was capable of selectively degrading BRD4 in cancer cells. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or in some cases inactivity. Further contrasting from cereblon or VHL-recruiting degradation, we show that BT1 treatment not only leads to BCR-ABL degradation, but also stabilizes the endogenous RNF114 substrate and tumor suppressor substrate p21. This leads to additional anti-proliferative effects in leukemia cancer cells beyond those observed with cereblon or VHL-recruiting BCR-ABL PROTACs. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs with unique additional benefits for oncology applications. We also further demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing and unpredictable selectivity for the degradation of neo-substrate proteins.

biochemistry↗

bioRxiv2020

The Genomic Formation of Human Populations in East Asia

The deep population history of East Asia remains poorly understood due to a lack of ancient DNA data and sparse sampling of present-day people. We report genome-wide data from 191 individuals from Mongolia, northern China, Taiwan, the Amur River Basin and Japan dating to 6000 BCE - 1000 CE, many from contexts never previously analyzed with ancient DNA. We also report 383 present-day individuals from 46 groups mostly from the Tibetan Plateau and southern China. We document how 6000-3600 BCE people of Mongolia and the Amur River Basin were from populations that expanded over Northeast Asia, likely dispersing the ancestors of Mongolic and Tungusic languages. In a time transect of 89 Mongolians, we reveal how Yamnaya steppe pastoralist spread from the west by 3300-2900 BCE in association with the Afanasievo culture, although we also document a boy buried in an Afanasievo barrow with ancestry entirely from local Mongolian hunter-gatherers, representing a unique case of someone of entirely non-Yamnaya ancestry interred in this way. The second spread of Yamnaya-derived ancestry came via groups that harbored about a third of their ancestry from European farmers, which nearly completely displaced unmixed Yamnaya-related lineages in Mongolia in the second millennium BCE, but did not replace Afanasievo lineages in western China where Afanasievo ancestry persisted, plausibly acting as the source of the early-splitting Tocharian branch of Indo-European languages. Analyzing 20 Yellow River Basin farmers dating to [~]3000 BCE, we document a population that was a plausible vector for the spread of Sino-Tibetan languages both to the Tibetan Plateau and to the central plain where they mixed with southern agriculturalists to form the ancestors of Han Chinese. We show that the individuals in a time transect of 52 ancient Taiwan individuals spanning at least 1400 BCE to 600 CE were consistent with being nearly direct descendants of Yangtze Valley first farmers who likely spread Austronesian, Tai-Kadai and Austroasiatic languages across Southeast and South Asia and mixing with the people they encountered, contributing to a four-fold reduction of genetic differentiation during the emergence of complex societies. We finally report data from Jomon hunter-gatherers from Japan who harbored one of the earliest splitting branches of East Eurasian variation, and show an affinity among Jomon, Amur River Basin, ancient Taiwan, and Austronesian-speakers, as expected for ancestry if they all had contributions from a Late Pleistocene coastal route migration to East Asia.

genomics↗

bioRxiv2020

Negative effects characterization and comparative transcriptomics elucidation on the lag phase of an industrial S. cerevisiae under the corn stover hydrolysate stress

During biofuels fermentation from pretreated lignocellulosic biomass, the strong toxicity of the lignocellulose hydrolysate is resulted from the synergistic effect of multiple lignocellulosic inhibitors, which far exceeds the sum of effects caused by every single inhibitor. Meanwhile, the synergistic effect is unclear and the underlying response mechanism of the industrial yeast towards the actual pretreated lignocellulose hydrolysate is still under exploration. Here, we employed an industrial S. cerevisiae for the transcriptomic analysis in two time points (early and late) of the lag phase under the corn stover hydrolysate stress. As investigation, the corn stover hydrolysate caused the accumulation of reactive oxygen species (ROS), damages of mitochondrial membrane and endoplasmic reticulum (ER) membrane in the industrial S. cerevisiae YBA_08 during the lag phase, especially these negative effects were more significant at the early lag phase. Based on the transcriptome profile, the industrial S. cerevisiae YBA_08 might recruit stress-related transcription factors (MSN4, STE12, SFL1, CIN5, COM2, MIG3, etc.) through the mitogen-activated protein kinase (MAPK)-signaling pathway to induce a transient G1/G2 arrest, and to activate defense bioprocesses like protectants metabolism, sulfur metabolism, glutaredoxin system, thioredoxin system, heat shock proteins chaperone and oxidoreductase detoxification, resisting those compounded stresses including oxidative stress, osmotic stress and structural stress. Surprisingly, this defense system might be accompanied with the transient repression of several bioprocesses like fatty acid metabolism, purine de novo biosynthesis and ergosterol biosynthesis. ImportanceThis research systematically demonstrated the lag phase response of an industrial yeast to the lignocellulosic hydrolysate in transcriptional level, providing a molecular fundament for understanding the synergistic effect of various lignocellulosic inhibitors and the regulatory mechanism of tolerance for industrial yeasts under this stress.

microbiology↗

bioRxiv2020

Reprogramming progressive cells display low CAG promoter activity

There is wide variability in the propensity of somatic cells to reprogram into pluripotency in response to the Yamanaka factors. How to segregate these variability to enrich for cells of specific traits that reprogram efficiently remains challenging. Here we report that the variability in reprogramming propensity is associated with the activity of the MKL1/SRF transcription factor and concurs with small cell size as well as rapid cell cycle. Reprogramming progressive cells can be prospectively identified by their low activity of a widely used synthetic promoter, CAG. CAGlow cells arise and expand during cell cycle acceleration in the early reprogramming culture of both mouse and human fibroblasts. Our work illustrate a molecular scenario underlying the distinct reprogramming propensities and demonstrate a convenient practical approach for their enrichment.

cell biology↗

bioRxiv2020

A Novel Non-hypermucoviscous ST11 Hypervirulent Carbapenem-resistant Klebsiella pneumoniae Lacking Classical Virulence Factors

BackgroundHypervirulent Klebsiella pneumoniae lacking classical virulence factors is uncommon, and the virulence mechanisms of this organism are not understood. MethodsFollowing a retrospective study of carbapenem-resistant K. pneumoniae based on core genome multilocus sequence typing (cgMLST), isolates that caused high mortality were investigated with a genome-wide association study (GWAS), proteome analysis and an animal model. ResultsThe sublineage of sequence type 11 (ST11) K. pneumoniae, which belongs to complex type 3176 (CT3176) and K-locus 47 (KL47), was highlighted due to the high mortality of infected patients. GWAS analysis showed that ampR was associated with the CT3176 isolates. In a mouse model, the mortality of ampR-carrying isolates was comparable to that of the typical hypervirulent isolate GM2. Even during the first 24 hours of infection, the bacterial load and pathological changes of the ampR-carrying isolates in the lungs were more severe than those of GM2. The ampR complement mutant was able to enhance the virulence of the KL47 isolate but not the virulence of KL1. Proteome analysis showed that the expression of WcaJ in the ampR+ isolates was significantly higher than that in the ampR- isolates, and this result was also confirmed by transcription tests and capsule staining. It is suggested that the enhancement of the initial stage of capsule synthesis may be the cause of the high virulence of these non-hypermucoviscous ST11 carbapenem-resistant K. pneumoniae isolates. ConclusionsNon-hypermucoviscous ST11 hypervirulent carbapenem-resistant K. pneumoniae warrants continued surveillance and investigation.

microbiology↗

bioRxiv2020

CasRx-mediated RNA targeting prevents choroidal neovascularization in a mouse model of age-related macular degeneration

The smallest Cas13 family protein, CasRx, has a high cleavage activity and targeting specificity, offering attractive opportunity for therapeutic applications. Here we report that delivery of CasRx by adeno-associated virus via intravitreal injection could efficiently knockdown Vegfa transcripts and significantly reduce the area of laser-induced choroidal neovascularization in a mouse model of age-related macular degeneration. Thus, RNA-targeting CRISPR system could be used for in vivo gene therapy.

genetics↗

bioRxiv2020

Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure in Toxoplasma gondii

Primary cilia are important organizing centers that control diverse cellular processes. Apicomplexan parasites like Toxoplasma gondii have a specialized cilium-like structure called the conoid that organizes the secretory and invasion machinery critical for the parasites lifestyle. The proteins that initiate the biogenesis of this structure are largely unknown. We identified the Toxoplasma ortholog of the conserved kinase ERK7 as essential to conoid assembly. Parasites in which ERK7 has been depleted lose their conoids late during maturation and are immotile and thus unable to invade new host cells. This is the most severe phenotype to conoid biogenesis yet reported, and is made more striking by the fact that ERK7 is not a conoid protein, as it localizes just basal to the structure. ERK7 has been recently implicated in ciliogenesis in metazoan cells, and our data suggest that this kinase has an ancient and central role in regulating ciliogenesis throughout Eukaryota.

cell biology↗

bioRxiv2020

Phytophthora infestans Ago1-bound miRNA promotes potato late blight disease

O_LIPhytophthora spp. incite serious plant damages by exploiting a large number of effector proteins and small RNAs (sRNAs). Several reports are describing modulation of host RNA biogenesis and defence gene expression. Here, we analysed P. infestans Argonaute (Ago) 1 associated small RNAs during potato leaf infection. C_LIO_LIsRNAs were co-immunoprecipitated, deep sequenced and analysed against the P. infestans and potato genomes, followed by transgenic and biochemical analyses on a predicted host target. C_LIO_LIExtensive targeting of potato and pathogen-derived sRNAs to a large number of mRNAs was observed, including 206 sequences coding for resistance (R) proteins in the host genome. The single miRNA encoded by P. infestans (miR8788) was found to target a potato lipase-like membrane protein-encoding gene (StLL1) localized to the tonoplast. Analyses of stable transgenic potato lines harbouring overexpressed StLL1 or artificial miRNA gene constructs demonstrated the importance of StLL1 during infection by P. infestans. Similarly, a miR8788 knock-down strain showed reduced growth on potato compared to the wild-type strain 88069. C_LIO_LIThe data suggest that sRNA encoded by P. infestans can affect potato mRNA and thereby promote disease. Knowledge of the impact of pathogen small RNAs in plant defence mechanisms is of major significance to succeed in improved disease control management. C_LI

plant biology↗

bioRxiv2019

Cross-validation of technologies for genotyping CYP2D6 and CYP2C19

BackgroundCYP2D6 and CYP2C19 are cytochrome P450 enzymes involved in the metabolism of many medications from multiple therapeutic classes. Associations between patterns of variants (known as haplotypes) in the genes encoding them (CYP2D6 and CYP2C19) and enzyme activities are well described. The genes in fact comprise 21% of biomarkers in drug labels. Despite this, genotyping is not common, partly attributable to its challenging nature (CYP2D6 having >100 haplotypes, including those with sequence from an adjacent pseudogene, and gene duplications). We cross-validated different methodologies for identifying haplotypes in these genes against each other. MethodsNinety-two samples with a variety of CYP2D6 and CYP2C19 genotypes according to prior AmpliChip CYP450 and TaqMan CYP2C19*17 data were selected from the Genome-based therapeutic drugs for depression (GENDEP) study. Genotyping was performed with TaqMan copy number variant (CNV) and single nucleotide variant (SNV) analysis, the next generation sequencing-based Ion S5 AmpliSeq Pharmacogenomics Panel, PharmacoScan, long-range polymerase chain reaction (L-PCR) followed by amplicon analysis, and Agena for CYP2C19. Variant pattern to haplotype translation was automated. ResultsThe inter-platform concordance for CYP2C19 was high (up to 100% for available data). For CYP2D6, the IonS5-PharmacoScan concordance was 94% for a range of variants tested apart from those with at least one extra copy of a CYP2D6 gene (occurring at a frequency of 3.8%, 33/853), or those with substantial sequence derived from pseudogene, known as hybrids (3%, 26/853). ConclusionsInter-platform concordance for CYP2C19 was high, and, moreover, the Ion S5 and PharmacoScan data were 100% concordant with that from a TaqMan CYP2C19*2 assay. We have also demonstrated feasibility of using an NGS platform for genotyping CYP2D6 and CYP2C19, with automated data interpretation methodology. This points the way to a method of making CYP2D6 and CYP2C19 genotyping more readily accessible.

genetics↗