Biology subjects

Peng, X.

Publications and source records attributed to Peng, X..

At least 127 records · Page 7Linked to original sources

bioRxiv2022

Excited-state dynamics of the all-trans protonated retinal Schiff base in CRABPII-based rhodopsin mimics

The rhodopsin mimic is a chemically synthetized complex with the retinyl Schiff base (RSB) formed between protein and the retinal chromophore, which can mimic the natural rhodopsin-like protein. The artificial rhodopsin mimic is more stable and designable than the natural protein and hence has wider uses in photon detection devices. The mimic structure RSB, like the case in the actual rhodopsin-like protein, undergoes isomerization and protonation throughout the photoreaction process. As a result, understanding the dynamics of the RSB in the photoreaction process is critical. In this study, the transient absorption spectra (TAS) of three mutants of the cellular retinoic acid-binding protein II (CRABPII)-based rhodopsin mimic at PH = 3 were recorded, from which the related excited-state dynamics of the all-trans protonated RSB (AT-PRSB) were investigated. The transient fluorescence spectra (TFS) measurements are used to validate some of the dynamic features. We find that the excited-state dynamics of AT-PRSB in three mutants share a similar pattern that differs significantly from the dynamics of 15-cis PRSB (15C-PRSB) of the rhodopsin mimic in neutral solution. By comparing the dynamics across the three mutants, we discovered that the aromatic residues near the {beta}-ionone ring structure of the retinal may help stabilize the AT-PRSB and hence slow down its isomerization rate. Furthermore, from the three mutants, we find one protein with near-infrared fluorescence emission up to 688 nm, leading to further possible applications in sensing or bioimaging.

biochemistry↗

bioRxiv2021

Miniature Structured Illumination Microscope for in vivo 3D Imaging of Brain Structures with Optical Sectioning

We present a high-resolution miniature, light-weight fluorescence microscope with electrowetting lens and onboard CMOS for high resolution volumetric imaging and structured illumination for rejection of out-of-focus and scattered light. The miniature microscope (SIMscope3D) delivers structured light using a coherent fiber bundle to obtain optical sectioning with an axial resolution of 18 m. Volumetric imaging of eGFP labeled cells in fixed mouse brain tissue at depths up to 220 m is demonstrated. The functionality of SIMscope3D to provide background free 3D imaging is shown by recording time series of microglia dynamics in awake mice at depths up to 120 m in the brain.

bioengineering↗

bioRxiv2021

In silico study on the effects of disulfide bonds in ORF8 of SARS-CoV-2

The COVID-19 epidemic, caused by virus SARS-CoV-2, has been a pandemic and threatening everyones health in the past two years. In SARS-CoV-2, the accessory protein ORF8 plays an important role in immune modulation. Here we present an in silico study on the effects of the disulfide bonds in ORF8, including the effects on the structures, the binding sites and free energy when ORF8 binds to the human leukocyte antigen (HLA-A). Using the explicit solvent Molecular Dynamics (MD) simulations, we collect the conformational ensembles on ORF8 with different disulfide bonds reduction schemes. With a new visualization technique on the local geometry, we analyze the effects of the disulfide bonds on the structure of ORF8. We find that the disulfide bonds have large influences on the loop regions of the surface. Moreover, by performing docking between HLA-A and the conformational ensembles of ORF8, we predict the preferred binding sites and find that most of them are little affected by the disulfide bonds.Further, we estimate the binding free energy between HLA-A and ORF8 with different disulfide bonds reductions. In the end, from the comparison with the available experimental results on the epitopes of ORF8, we validated our binding sites prediction. All the above observations may provide inspirations on inhibitor/drug design against ORF8 based on the binding pathway with HLA-A.

biophysics↗

bioRxiv2021

Replication protein Rep provides selective advantage to viruses in the presence of CRISPR-Cas immunity

Prokaryotic viruses express anti-CRISPR (Acr) proteins to inhibit the host adaptive immune system, CRISPR-Cas. While the virus infection biology was shown to be strongly dependent on the relative strengths of the host CRISPR-Cas and viral Acrs, little is known about the role of the core processes of viral life cycle (replication, packaging etc) in defence/anti-defence arms race. Here, we demonstrate the selective advantage provided by a replication initiator, Rep, in the context of CRISPR-Acr interactions. First, we developed a two-host based CRISPR-Cas genome editing tool for the deletion of highly conserved and thus potentially important viral genes. Using this strategy, we deleted a highly conserved Rep-coding gene, gp16, from the genome of Sulfolobus islandicus rod-shaped virus 2 (SIRV2). The knockout mutant ({Delta}gp16) produced around 4 fold less virus in a CRISPR-null host, suggesting that Rep is the major contributor to replication initiation in Rudiviridae. Indeed, DNA sequencing revealed Rep-dependent replication initiation from the viral genome termini, in addition to Rep-independent replication initiation from non-terminal sites. Intriguingly, the lack of Rep showed a profound effect on virus propagation in a host carrying CRISPR-Cas immunity. Accordingly, the co-infecting parental virus (rep-containing) outcompeted the {Delta}gp16 mutant much more quickly in CRISPR-containing host than in CRISPR-null host, demonstrating a selective advantage provided by Rep in the presence of host CRISPR-Cas immunity. Despite the non-essentiality, rep is carried by all known members of Rudiviridae, which is likely an evolutionary outcome driven by the ubiquitous presence of CRISPR-Cas in Sulfolobales. ImportanceCRISPR-Cas and anti-CRISPR proteins are accessary to prokaryotes and their viruses respectively. To date, research has been focused on their diversity, molecular mechanisms and application in genome editing. How CRISPR-Acr arms race influence the evolution of viral core genes involved in the basic virus life cycle remained a gap of knowledge so far. This study provides the first evidence that CRISPR-Acr arms race poses a selection pressure on the efficiency of viral genome replication, forcing viruses to evolve highly productive replication machineries..

microbiology↗

bioRxiv2021

An ex vivo culture model of kidney podocyte injury reveals mechanosensitive, synaptopodin-templating, sarcomere-like structures.

Chronic kidney diseases are widespread and incurable. The biophysical mechanisms underlying them are unclear, in part because material systems for reconstituting the microenvironment of the relevant kidney cells are limited. A critical question is how kidney podocytes (glomerular epithelial cells) regenerate the foot processes of the filtration apparatus following injury. Recently identified sarcomere-like structures (SLSs) with periodically spaced myosin IIA (a contractile protein) and synaptopodin (an actin-associated protein) appear in injured podocytes in vivo. We hypothesized that SLSs template synaptopodin in the initial stages of recovery, and tested this hypothesis by developing an ex vivo culture system that models both kidney physiology and pathophysiology. SLSs were observed in vitro for the first time as podocytes migrated out of harvested kidney glomeruli onto micropatterns of physiologically relevant proteins. SLSs emerged over two days, and cells formed foot process-like extensions from these periodically spaced proteins. SLS distributions and morphology were sensitive to actomyosin inhibitors, substrate stiffness, and extracellular matrix proteins associated with pathology. These results indicate a role for mechanobiological factors in podocyte recovery from injury, and suggest SLSs as a target for therapeutic intervention.

cell biology↗

bioRxiv2021

Vagus nerve stimulation accelerates motor learning through cholinergic modulation

Vagus nerve stimulation (VNS) is a neuromodulation therapy for a broad and rapidly expanding set of neurologic conditions. Classically used to treat epilepsy and depression, VNS has recently received FDA approval for stroke rehabilitation and is under preclinical and clinical investigation for other neurologic indications. Despite benefits across a diverse range of neurological disorders, the mechanism through which VNS influences central nervous system circuitry is not well described, limiting therapeutic optimization. A deeper understanding of the influence of VNS on neural circuits and activity is needed to maximize the use of VNS therapy across a broad range of neurologic conditions. To investigate how VNS can influence the neurons and circuits that underlie behavior, we paired VNS with upper limb movement in mice learning a skilled motor task. We leveraged genetic tools to perform optogenetic circuit dissection, as well as longitudinal in vivo imaging of calcium activity in cortical neurons to understand the effect of VNS on neural function. We found that VNS robustly enhanced motor learning when temporally paired with successful movement outcome, while randomly applied VNS impaired learning. This suggests that temporally-precise VNS may act through augmenting outcome cues, such as reinforcement signals. Within motor cortex, VNS paired with movement outcome selectively modulates the neural population that represents outcome, but not other movement-related neurons, across both acute and behaviorally-relevant timescales. Phasic cholinergic signaling from basal forebrain is required both for VNS-driven improvements in motor learning and the effects on neural activity in M1. These results indicate that VNS enhances motor learning through precisely-timed phasic cholinergic signaling to reinforce outcome, resulting in the recruitment of specific, behaviorally-relevant cortical circuits. A deeper understanding of the mechanisms of VNS on neurons, circuits and behavior provides new opportunities to optimize VNS to treat neurologic conditions.

neuroscience↗

bioRxiv2021

Anatomical and functional connectivity support the existence of a salience network node within the caudal ventrolateral prefrontal cortex

Three large-scale brain networks are considered essential to cognitive flexibility: the ventral and dorsal attention (VAN and DAN) and salience (SN) networks. The ventrolateral prefrontal cortex (vlPFC) is a known component of the VAN and DAN, but there is an important gap in the current knowledge regarding its involvement in the SN. In this study, we used a translational and multimodal approach to fulfill this gap and demonstrate the existence of a SN node within the vlPFC. First, we used tract-tracing methods in non-human primates (NHP) to quantify the anatomic connectivity strength between the different vlPFC areas and the frontal and insular cortices. The strongest connections with the dorsal anterior cingulate cortex (dACC) and anterior insula (AI) locations comprising the two main cortical SN nodes were derived from the caudal area 47/12. This location also has strong axonal projections to subcortical structures of the salience network, including the dorsomedial thalamus, hypothalamus, sublenticular extended amygdala, and periaqueductal gray. Second, we used a seed-based functional connectivity analysis in NHP resting-state functional MRI (rsfMRI) data to validate the caudal area 47/12 as an SN node. Third, we used the same approach in human rsfMRI data to identify a homologous structure in caudal area 47/12, also showing strong connections with the SN cortical nodes, thus confirming the caudal area 47/12 as the SN node in the vlPFC. Taken together, the vlPFC contains nodes for all three cognitive networks, the VAN, DAN, and SN. Thus, the vlPFC is in a position to switch between these three cognitive networks, pointing to a key role as an attentional hub. Its tight additional connections to the orbitofrontal, dorsolateral, and ventral premotor cortices, places the vlPFC at the center for switching behaviors based on environmental stimuli, computing value and cognitive control.

neuroscience↗

bioRxiv2021

Attention-based deep multiple instances learning for classifying circular RNA and other long non-coding RNA

Circular RNA (circRNA) is a distinguishable circular formed long non-coding RNA (lncRNA), which has specific roles in transcriptional regulation, multiple biological processes. The identification of circRNA from other lncRNA is necessary for relevant research. In this study, we designed attention-based multi-instance learning (MIL) network architecture, which can be fed with raw sequence, to learn the sparse features in sequences and accomplish the identification task for circRNAs. The model outperformed previously reported models. Following the effectiveness validation of the attention score by the handwritten digit dataset, the key sequence loci underlying circRNAs recognition were obtained based on the corresponding attention score. Moreover, the motif enrichment analysis of the extracted key sequences identified some of the key motifs for circRNA formation. In conclusion, we designed a deep learning network architecture suitable for gene sequence learning with sparse features and implemented to the circRNA identification, and the network has a strong representation capability with its indication of some key loci.

genomics↗

bioRxiv2021

Single cell based high-throughput Ig and TCR repertoire sequencing analysis in rhesus macaques

Recent advancements in microfluidics and high-throughput sequencing technologies have enabled recovery of paired heavy- and light-chains of immunoglobulins (Ig) and VDJ- and VJ-chains of T cell receptors (TCR) from thousands of single cells simultaneously in humans and mice. Despite rhesus macaques being one of the most well-studied model organisms for the human adaptive immune response, high-throughput single cell immune repertoire sequencing assays are not yet available due to the complexity of these polyclonal receptors. Here we employed custom primers that capture all known rhesus macaque Ig and TCR isotypes and chains that are fully compatible with a commercial solution for single cell immune repertoire profiling. Using these rhesus specific assays, we sequenced Ig and TCR repertoires in over 60,000 cells from cryopreserved rhesus PBMC, splenocytes, and FACS-sorted B and T cells. We were able to recover every Ig isotype and TCR chain, measure clonal expansion in proliferating T cells, and pair Ig and TCR repertoires with gene expression profiles of the same single cells. Our results establish the ability to perform high-throughput immune repertoire analysis in rhesus macaques at the single cell level.

immunology↗

bioRxiv2021

TMT-based proteomic analysis of the antibiotic effects of ShangKeHuangShui against Straphylococcus aureus.

Post-traumatic infection is a serious orthopedic trauma complication commonly caused by Staphylococcus aureus (SAU). ShangKeHuangShui, also known as Yellow Aqueous Concentrate of Traumatology Herbs (YACTH), is prepared from six Chinese herbal medicines, which has been used for decades in our hospital to prevent post-traumatic infection. In the present study, we investigated the in vitro antibacterial effects and underlying mechanism of YACTH against SAU. YACTH exhibited significant antibacterial activity against SAU with the minimum inhibitory concentration (MIC) of 8.625 mg/mL. Proteomic analysis based on Tandem mass tag (TMT) showed different protein expression levels in SAU under the YACTH and control conditions. Compared to the control group, the expression level of 490 proteins of YACTH treated group significantly changed (>1.2 fold, P <0.05). Biological informatics analysis showed that these differential proteins were widely involved in a variety of biological processes and multiple metabolic pathways. We then selected 26 target proteins to further conduct Parallel reaction monitoring (PRM). The results revealed that the 9 down-regulated proteins were mainly involved in RNA polymerase, oxidative phosphorylation, ABC transporter and glycolysis; whereas, the 17 up-regulated proteins are related to RNA degradation, mismatch repair and ribosome. In conclusion, YACTH, which contains the water-soluble components of six Chinese herbs, is first reported to exhibit antibacterial activity against SAU in vitro. The results provide novel insights into the antibacterial mechanisms of YACTH on protein networks, which may help us find new potential antibiotic targets.

microbiology↗

bioRxiv2021

WDR31 is a novel ciliopathy protein displaying functional redundancy with GTPase-activating proteins ELMOD and RP2 in recruiting BBSome to cilium

The correct intraflagellar transport (IFT) assembly at the ciliary base and the IFT turnaround at the ciliary tip are key for the IFT to perform its function, but we still have poor understanding about how these processes are regulated. Here, we identify WDR31 as a new ciliary protein, and analysis from zebrafish and Caenorhabditis elegans reveals the role of WDR31 in regulating the cilia morphology. We find that loss of WDR-31 together with RP-2 and ELMD-1 (the sole ortholog ELMOD1-3) results in ciliary accumulations of IFT Complex B components and KIF17 kinesin, with fewer IFT/BBSome particles traveling along cilia in both anterograde and retrograde directions, suggesting that the IFT/BBSome entry into cilia and exit from cilia are impacted. Furthermore, anterograde IFT in the middle segment travel at increased speed in wdr-31;rpi-2;elmd-1. Remarkably, a non-ciliary protein leaks into cilia of wdr-31;rpi-2;elmd-1 possible due to IFT defects. This work reveals WDR31-RP-2-ELMD-1 as IFT and BBSome trafficking regulators.

genetics↗

bioRxiv2021

Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome

SARS-CoV-2, as the causation of severe epidemic of COVID-19, is one kind of positive single-stranded RNA virus with high transmissibility. However, whether or not SARS-CoV-2 can integrate into host genome needs thorough investigation. Here, we performed both RNA sequencing (RNA-seq) and whole genome sequencing on SARS-CoV-2 infected human and monkey cells, and investigated the presence of host-virus chimeric events. Through RNA-seq, we did detect the chimeric host-virus reads in the infected cells. But further analysis using mixed libraries of infected cells and uninfected zebrafish embryos demonstrated that these reads are falsely generated during library construction. In support, whole genome sequencing also didnt identify the existence of chimeric reads in their corresponding regions. Therefore, the evidence for SARS-CoV-2s integration into host genome is lacking. One-Sentence SummarySARS-CoV-2 does not integrate into host genome through whole genome sequencing.

genomics↗

bioRxiv2021

Genome of the giant panda roundworm illuminates its host shift and parasitic adaptation

Baylisascaris schroederi, a bamboo-feeding giant panda (Ailuropoda melanoleuca)-specific roundworm (ascaridoid) parasite, is the causative agent of baylisascariasis, which represents a leading reason for the mortality of wild giant panda populations and therefore poses a significant threat to giant panda conservation. Here we present a 293-Mb chromosome-level genome assembly of B. schroederi to inform its biology, including host adaptations. Comparative genomics revealed an evolutionary trajectory accompanied by host-shift events in ascaridoid parasite lineages after host separations, suggesting their potential transmissions and fast adaptations to hosts. Genomic and anatomical lines of evidence, including expansion and positive selection of genes related to cuticle and basal metabolisms, indicates that B. schroederi undergoes specific adaptations to survive in the sharp-edged bamboo enriched gut of giant panda by structurally increasing its cuticle thickness and efficiently utilizing host nutrients during gut parasitism. Also, we characterized the secretome and predicted potential drug and vaccine targets for new interventions. Overall, this genome resource provides new insights into the host adaptation of B. schroederi to giant panda as well as the host-shift events in ascaridoid parasite lineages. These findings also add to our knowledge on the unique biology of the giant panda roundworm and aid the development of much-needed novel strategies for the control of baylisascariasis and thus the protection of giant panda populations.

genomics↗

bioRxiv2021

Lipid Bilayer Induces Contraction of the Denatured State Ensemble of a Helical-Bundle Membrane Protein

Defining the denatured state ensemble (DSE) and intrinsically disordered proteins is essential to understanding protein folding, chaperone action, degradation, translocation and cell signaling. While a majority of studies have focused on water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we reconstituted the DSE of a helical-bundle membrane protein GlpG of Escherichia coli in native lipid bilayers and measured the DSEs conformation and compactness. The DSE was obtained using steric trapping, which couples spontaneous denaturation of a doubly biotinylated GlpG to binding of two bulky monovalent streptavidin molecules. Using limited proteolysis and mass spectrometry, we mapped the flexible regions in the DSE. Using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy, we determined the dimensions of the DSE. Finally, we employed our Upside model for molecular dynamics simulations to generate the DSE including the collapsed and fully expanded states in a bilayer. We find that the DSE is highly dynamic involving the topology changes of transmembrane segments and their unfolding. The DSE is expanded relative to the native state, but only to 55-90% of the fully expanded condition. The degree of expansion depends on the chemical potential with regards to local packing and the lipid composition. Our result suggests that the E. colis native lipid bilayer promotes the association of helices in the DSE of membrane proteins and, probably in general, facilitating interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes. SignificanceHere, we delineate the conformation of the denatured state ensemble (DSE) of a membrane protein confined in a native lipid bilayer and assay whether the bilayer permits full expansion or nonspecific collapse of the DSE. Using the intramembrane protease GlpG as a model, we find that the denatured state is a dynamic ensemble involving topological changes and local unfolding of transmembrane segments. The bilayer tends to contract the DSE relative to the fully lipid-solvated, expanded conformations while the degree of compactness is determined by the balance between protein-lipid, lipid-lipid and protein-protein interactions. These findings provide new insights into the lipid bilayer as a solvent that mediates folding, chaperone action, turnover and protein-protein interactions in cell membranes.

biophysics↗

bioRxiv2021

Imaging the ultrastructures and dynamics of live erythrocyte membranes at the single-molecule level with a far-red probe on a microfluidic platform

Both the ultrastructures and dynamics of living erythrocyte membranes provide critical criteria for clinical diagnostics. However, it is challenging to simultaneously visualize these features at the single-molecule level due to the rigid photophysical requirements of different single-molecule imaging techniques. Herein, we rationally developed a far-red boron dipyrromethene membrane (BDP-Mem) probe that not only retained consistent and intensive single-molecule emission but also possessed the capability to photoswitch on cellular membranes. We also constructed a microfluidic platform for the noninvasive trapping and long-term imaging of nonadherent erythrocytes. By combining these advantageous technologies, super-resolution reconstruction and single-molecule tracking of living human RBC membranes were achieved at the molecular scale in a high-throughput fashion. Our integrated paradigm defines a quantitative approach for analyzing living RBC membranes under physiological and pathological conditions, improving imaging precisions and revealing new perspectives for future disease diagnostic approaches.

biophysics↗

bioRxiv2021

CoVac501, a self-adjuvanting peptide vaccine conjugated with TLR7 agonists, against SARS-CoV-2 induces protective immunity

Safe, economical and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to achieve adequate herd immunity and halt the pandemic. We have constructed a novel SARS-CoV-2 vaccine, CoVac501, which is a self-adjuvanting peptide vaccine conjugated with Toll-like receptor 7 (TLR7) agonists. The vaccine contains two immunodominant peptides screened from receptor-binding domain (RBD) and is fully chemically synthesized. And the vaccine has optimized nanoemulsion formulation, outstanding stability and safety. In non-human primates (NHPs), CoVac501 elicited high and persistent titers of RBD-specific and protective neutralizing antibodies (NAbs), which were also effective to RBD mutations. CoVac501 was found to elicit the increase of memory T cells, antigen-specific CD8+ T cell responses and Th1-biased CD4+ T cell immune responses in NHPs. More importantly, the sera from the immunized NHPs can prevent infection of live SARS-CoV-2 in vitro. One-Sentence SummaryA novel SARS-CoV-2 vaccine we developed, CoVac501, which is a fully chemically synthesized and self-adjuvanting peptides conjugated with TLR7 agonists, can induce high-efficient humoral and cellular immune responses against SARS-CoV-2.

immunology↗

bioRxiv2021

Deacetylation of IFIT2 mediated by HDAC5 promotes the stemness and progression of glioma

BackgroundGlioma is the most common primary brain tumor, and the tumor stemness is a major regulatory factor affecting the progression, metastasis and recurrence of glioma. Recent research has shown that, nonhistone acetylation is widely involved in key cellular processes, including stemness regulation. The deacetylase inhibitors are promising new drugs, but their application and molecular mechanism in glioma have not been elucidated. MethodsCCK8 and colony formation assay were used to detect cell proliferation, transwell assay was used to detect cell migration, flow cytometry was used to analyze cell apoptosis, sphere formation assay and western blot were used to detect the status of stemness. RNA-sequence, quantitative PCR and western blot were performed to screen the key molecules mediating LBH589 function. Immunoprecipitation (IP) and western blot were used to analyze the acetylation level of IFIT2. The SiRNA target HDAC4 or HDAC5, overexpression plasmids of acetyltransferases were used to identify the acetyltransferase and deacetylase regulating IFIT2. The regulatory mechanism was explored by IP and ubiquitination analysis. Finally, the xenograft tumor model in nude mice was constructed and further analyzed in vivo. ResultsThe data showed that IFIT2 mediates the HDACi LBH589 inhibition on cell proliferation, migration and stemness, and contribution to autophagy and apoptosis in glioma. And the down-regulation of IFIT2 in glioma was confirmed to be related to its deacetylation by overexpression HDAC5, which promotes the stemness and progression of glioma. Further, deacetylation of IFIT2 by HDAC5 was demonstrated to induce its ubiquitination and subsequent protein instability, which led to loss of anti-tumor activity for IFIT2, and acceleration to glioma stemness and progression. In addition, the results indicated that IFIT2 inhibits PKC pathway, and suppressing of IFIT2 promotes tumor growth in vivo. ConclusionsThese results not only clarify a novel post-transcriptional regulatory mode of IFIT2, but also provide a new sight of molecular mechanism for HDACi in glioma.

cancer biology↗

bioRxiv2021

Single-cell transcriptome profiling simulation reveals the impact of sequencing parameters and algorithms on clustering

Despite of scRNA-seq analytic algorithms developed, their performance for cell clustering cannot be quantified due to the unknown "true" clusters. Referencing the transcriptomic heterogeneity of cell clusters, a "true" mRNA number matrix of cell individuals was defined as ground truth. Based on the matrix and real data generation procedure, a simulation program (SSCRNA) for raw data was developed. Subsequently, the consistence between simulated data and real data was evaluated. Furthermore, the impact of sequencing depth, and algorithms for analyses on cluster accuracy was quantified. As a result, the simulation result is highly consistent with that of the real data. It is found that mis-classification rate can be attributed to multiple reasons on current scRNA platforms, and clustering accuracy is not only sensitive to sequencing depth increasement, but can also be reflected by the position of the cluster on TSNE plot. Among the clustering algorithms, Gaussian normalization method is more appropriate for current workflows. In the clustering algorithms, k-means&louvain clustering method performs better in dimension reduced data than full data, while k-means clustering method is stable under both situations. In conclusion, the scRNA simulation algorithm developed restores the real data generation process, discovered impact of parameters on mis-clustering, compared the normalization/clustering algorithms and provided novel insight into scRNA analyses.

bioinformatics↗