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Background Massively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms. Results Here, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGI’s sequencers utilize a unique DNA nanoball (DNB) technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001 to 0.0004% under recommended procedures. Conclusions Single indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

To unravel the source of SARS-CoV-2 introduction and the pattern of its spreading and evolution in the United Arab Emirates, we conducted meta-transcriptome sequencing of 1067 nasopharyngeal swab samples collected between May 9th and Jun 29th, 2020 during the first peak of the local COVID-19 epidemic. We identified global clade distribution and eleven novel genetic variants that were almost absent in the rest of the world and that defined five subclades specific to the UAE viral population. Cross-settlement human-to-human transmission was related to the local business activity. Perhaps surprisingly, at least 5% of the population were co-infected by SARS-CoV-2 of multiple clades within the same host. We also discovered an enrichment of cytosine-to-uracil mutation among the viral population collected from the nasopharynx, that is different from the adenosine-to-inosine change previously reported in the bronchoalveolar lavage fluid samples and a previously unidentified upregulation of APOBEC4 expression in nasopharynx among infected patients, indicating the innate immune host response mediated by ADAR and APOBEC gene families could be tissue-specific. The genomic epidemiological and molecular biological knowledge reported here provides new insights for the SARS-CoV-2 evolution and transmission and points out future direction on host–pathogen interaction investigation.

In order to overcome disturbances such as the instability of internal parameters or the actuator fault, the time-varying proportional-integral sliding-mode surface is defined for coordinated control of the excitation generator and the steam valve of waste heat power generation units, and a controller based on sliding-mode function is designed which makes the system stable for a limited time and gives it good performance. Based on this, a corresponding fault estimation law is designed for specific faults of systems, and a sliding-mode fault-tolerant controller is constructed based on the fixed-time control theory so that the systems can still operate stably when an actuator fault occurs and have acceptable performance. The simulation results show that the tracking error asymptotically tends to be zero, and the fixed-time sliding-mode fault-tolerant controller can obviously improve the dynamic performance of the system.

Porcine rotavirus (PoRV) and porcine epidemic diarrhea virus (PEDV) usually co-infect pigs in modern large-scale piggery, which both can cause severe diarrhea in newborn piglets and lead to significant economic losses to the pig industry. The VP7 protein is the main coat protein of PoRV, and the S protein is the main structural protein of PEDV, which are capable of inducing neutralizing antibodies in vivo. In this study, a DNA vaccine pPI-2.EGFP.VP7.S co-expressing VP7 protein of PoRV and S protein of PEDV was constructed. Six 8-week-old mice were immunized with the recombinant plasmid pPI-2.EGFP.VP7.S. The high humoral immune responses (virus specific antibody) and cellular immune responses (IFN-γ, IL-4, and spleen lymphocyte proliferation) were evaluated. The immune effect through intramuscular injection increased with plasmid dose when compared with subcutaneous injection. The immune-enhancing effect of IFN-α adjuvant was excellent compared with pig spleen transfer factor and IL-12 adjuvant. These results demonstrated that pPI-2.EGFP.VP7.S possess the immunological functions of the VP7 proteins of PoRV and S proteins of PEDV, indicating that pPI-2.EGFP.VP7.S is a candidate vaccine for porcine rotaviral infection (PoR) and porcine epidemic diarrhea (PED).

Abstract Background Systematic errors can be introduced from DNA amplification during massively parallel sequencing (MPS) library preparation and sequencing array formation. Polymerase chain reaction (PCR)-free genomic library preparation methods were previously shown to improve whole genome sequencing (WGS) quality on the Illumina platform, especially in calling insertions and deletions (InDels). We hypothesized that substantial InDel errors continue to be introduced by the remaining PCR step of DNA cluster generation. In addition to library preparation and sequencing, data analysis methods are also important for the accuracy of the output data.In recent years, several machine learning variant calling pipelines have emerged, which can correct the systematic errors from MPS and improve the data performance of variant calling. Results Here, PCR-free libraries were sequenced on the PCR-free DNBSEQ™ arrays from MGI Tech Co., Ltd. (referred to as MGI) to accomplish the first true PCR-free WGS which the whole process is truly not only PCR-free during library preparation but also PCR-free during sequencing. We demonstrated that PCR-based WGS libraries have significantly (about 5 times) more InDel errors than PCR-free libraries.Furthermore, PCR-free WGS libraries sequenced on the PCR-free DNBSEQ™ platform have up to 55% less InDel errors compared to the NovaSeq platform, confirming that DNA clusters contain PCR-generated errors.In addition, low coverage bias and less than 1% read duplication rate was reproducibly obtained in DNBSEQ™ PCR-free using either ultrasonic or enzymatic DNA fragmentation MGI kits combined with MGISEQ-2000. Meanwhile, variant calling performance (single-nucleotide polymorphisms (SNPs) F-score>99.94%, InDels F-score>99.6%) exceeded widely accepted standards using machine learning (ML) methods (DeepVariant or DNAscope). Conclusions Enabled by the new PCR-free library preparation kits, ultra high-thoughput PCR-free sequencers and ML-based variant calling, true PCR-free DNBSEQ™ WGS provides a powerful solution for improving WGS accuracy while reducing cost and analysis time, thus facilitating future precision medicine, cohort studies, and large population genome projects. Competing Interest Statement The authors have no competing interests，besides some employees of MGI Tech Co., Ltd., BGI-Shenzhen and Complete Genomics Inc. have stock holdings in BGI. Footnotes * 1.The paper title was revised. 2.The abstract format and several descriptions were revised. 3.Parts of the authors' affiliated organization and available e-mail addresses were revised. 4.The competing interests were updated. 5.The paper content format is revised accroding to the requriments from the publication that plan to submit. * https://db.cngb.org/cnsa * List of abbreviations BWA Burrows-Wheeler aligner CNN model convolutional neural network model CNV Copy Number Variation DNBs DNA nanoballs FP false positive FN false negative GATK Genome Analysis Toolkit GIAB genome in a bottle hom-ref homozygous reference het heterozygous hom-alt homozygous alternate InDels insertions and deletions LFR long fragment read MPS massively parallel sequencing ML machine learning NGS next-generation sequencing PCR Polymerase chain reaction Pair-HMM read-likelihood calculation rWGS rapid whole-genome sequencing SNPs single-nucleotide polymorphisms ssDNA single-stranded DNA SPRI solid-phase reversible immobilization SV Structural Variation VQSR Variant quality score recalibration WGS whole genome sequencing WES whole-exome sequencing

Accurate next generation sequencing (NGS) is critical for understanding genetic predisposition to human disease and thus aiding clinical diagnosis and personalized precision medicine. Recent breakthroughs in massively parallel sequencing, especially when coupled with sample multiplexing, have driven sequencing cost down and made clinical genetic tests broadly affordable. However, intractable index mis-assignment (commonly exceeds 1%) has been reported on some widely used sequencing platforms. Burdensome unique dual indexing is now used to reduce this problem. Here, we investigated this quality issue on BGI sequencers using three major library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGI sequencers utilize a unique DNA nanoball (DNB) technology that is based on rolling circle replication (RCR) for array preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrate here that single index mis-assignment from free indexed oligos on these sequencers occurs at a rate of only one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying, as expected for the RCR process. Furthermore, the DNB-based NGS applications have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001% to 0.0004% using only single indexing. Therefore, single indexing with DNB sequencing technology provides a simple but effective method for sensitive research and clinical genetic assays that require the detection of low abundance sequences in a large number of samples.

The diversity of disease presentations warrants one single assay for detection and delineation of various genomic disorders. Herein, we describe a gel-free and biotin-capture-free mate-pair method through coupling Controlled Polymerizations by Adapter-Ligation (CP-AL). We first demonstrated the feasibility and ease-of-use in monitoring DNA nick-translation and primer extension by limiting the nucleotide input. By coupling these two controlled polymerizations by a reported non-conventional adapter ligation reaction 3' branch ligation, we evidenced that CP-AL significantly increased DNA-circularization efficiency (by 4-fold) and was applicable for different sequencing methods but at a faction of current cost. Its advantages were further demonstrated by fully elimination of small-insert-contaminated (by 39.3-fold) with a ~50% increment of physical coverage, and producing uniform genome/exome coverage and the lowest chimeric rate. It achieved single-nucleotide variants detection with sensitivity and specificity up to 97.3 and 99.7%, respectively, compared with data from small-insert libraries. In addition, this method can provide a comprehensive delineation of structural rearrangements, evidenced by a potential diagnosis in a patient with oligo-atheno-terato-spermia. Moreover, it enables accurate mutation identification by integration of genomic variants from different aberration types. Overall, it provides a potential single-integrated solution for detecting various genomic variants, facilitating a genetic diagnosis in human diseases. Footnotes * https://db.cngb.org/cnsa

Pd-based nanomaterials have shown great promise as potential mimic enzymes, but conventional catalysts use only a small fraction of the Pd content that located on the catalyst’s surface. Herein, we demonstrated that maximum atom utilization could be achieved by using single-atom Pd catalysts as oxidase mimic. The single-atom Pd nanozymes exhibit significantly enhanced catalytic efficiency, with a catalytic rate constant ( K cat ) and the catalytic efficiency ( K cat / K m ) values more than 625 and 4,837 times higher than those of horseradish peroxidase, respectively. A combined experimental and theoretical calculation reveals reactive oxygen species involved catalytic mechanism which endows single-atom Pd catalysts with excellent colorimetric analysis performance. Benefiting from the maximum atom utilization efficiency and well-defined structural features, the single-atom Pd nanozymes could be successfully applied for the total antioxidant capacity of fruit, determining the serum acid phosphatase activity as well as constructing NAND logic gate. This finding not only provides an effective strategy to maximize the noble-metal atom utilization efficiency as enzyme mimics, but also provides a new idea for extending their possible applications.

Skin cutaneous malignant melanoma (SKCM) is among the most aggressive forms of skin cancer, notorious for its rapid progression and poor prognosis under late diagnosis. This study investigates the role of circadian rhythm-related genes (CRGs) in SKCM addressing a gap in understanding how CRGs affect tumor progression and patient outcomes. An analysis of CRGs expression was conducted on SKCM samples derived from The Cancer Genome Atlas datasets(TCGA). Moreover, a correlation between various subtypes and their clinical features was identified. The study employed various bioinformatics methods, including differential expression analysis, consensus clustering, and survival analysis, to investigate the role of CRGs. The functional consequences of various CRG expression patterns were further investigated using immune infiltration analysis and gene set variation analysis (GSVA). A scoring system based on CRGs was developed to predict overall survival (OS) and treatment responses in SKCM patients. The predictive accuracy of this score system was then tested, and a nomogram was used to improve its clinical usefulness. Key findings from this study include significant genetic alterations in circadian rhythm-related genes (CRGs) in skin cutaneous melanoma (SKCM), such as mutations and CNVs. Two molecular subtypes with distinct clinical outcomes and immune profiles were identified. A prognostic model based on six CRGs ( , , , , , and ) was developed and validated with TCGA and GEO datasets, showing high predictive accuracy for overall survival (OS). A high CRGs score correlated with poor OS, immune checkpoint expression, and reduced sensitivity to several chemotherapeutics, including AKT inhibitor VIII and Camptothecin. This work provides valuable insights into the circadian regulation of SKCM and underscores the potential of CRGs as biomarkers for prognosis and targets for therapeutic interventions. The novel molecular subtypes and CRGs prognostic scoring model introduced in this study offer significant contributions to the understanding and management of SKCM.