Accurate Diagnosis of SARS-CoV-2 JN.1 by Sanger Sequencing of Receptor-Binding Domain Is Needed for Clinical Evaluation of Its Immune Evasion

Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used SGTF of TaqPath COVID-19 Combo Kit RT-qPCR as proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥ 6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing instead of proxy indicator to diagnose JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The nested PCR amplicons were sequenced. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed R203K, G204R and Q229K, the 3 mutations associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 of the 26 known amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: Sanger sequencing of a 445-base segment of the SARS-CoV-2 RBD is useful for accurate determination of emerging variants. The CDC may consider using Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.

AutoSeqMan:batch assembly of contigs for Sanger sequences

With the wide application of DNA sequencing technology, DNA sequences are still increasingly generated through the Sanger sequencing platform. SeqMan （in the LaserGene package） is an excellent program with an easy-to-use graphical user interface （GUI） employed to assemble Sanger sequences into contigs. However, with increasing data size, larger sample sets and more sequenced loci make contig assemble complicated due to the considerable number of manual operations required to run SeqMan. Here, we present the ＇autoSeqMan＇ software program, which can automatedly assemble contigs using SeqMan scripting language. There are two main modules available, namely, ＇Classification＇ and ＇Assembly＇. Classification first undertakes preprocessing work, whereas Assembly generates a SeqMan script to consecutively assemble contigs for the classified files. Through comparison with manual operation, we showed that autoSeqMan saved substantial time in the preprocessing and assembly of Sanger sequences. We hope this tool will be useful for those with large sample sets to analyze, but with little programming experience. It is freely available at https：//github.com/ Sun-Yanbo/autoSeqMan.

Cloning and Sequence Analysis of 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo

[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.

2021年老挝M_(S)6.0地震序列研究

云南地震活动与周边强震存在“构造相连,动力同源”的特征,研究周边强震的序列演化特征及发震构造,对云南地区地震研究具有重要意义。2021年12月24日老挝M_(S)6.0地震发生在滇西南地区的NW向整董断裂附近,震源机制解显示,此次地震是一次走滑型破裂事件,破裂方向与区域构造特征一致。老挝M_(S)6.0地震序列属于前震-主震-余震型序列,主震前震中附近出现3~4级地震非常活跃的现象,前震序列参数计算显示b值波动相对幅度较大,h值出现“上翘”形态,而余震序列b值和h值变化均相对平稳,主震的同震库伦应力结果表明老挝地震可能对云南地区有应力加载作用。

基于Sanger测序技术的云南荔枝园蠹虫种类初步鉴定

荔枝作为云南乡村振兴重要产业之一,随着面积的不断扩大,其种植生态环境多样化,病虫害的种类也随之增加。近几年在云南荔枝主要产区陆续发现蠹虫为害,调查荔枝蠹虫为害情况并鉴定其种类,对云南荔枝病虫害防治具有重要意义。采用5点取样法,针对云南荔枝5个主要产区蠹虫为害程度以及为害状进行调查,利用Sanger测序技术进行生理小种初步鉴定。结果发现,5个产区均有不同程度蠹虫为害,为害状与长蠹科昆虫为害状相似。经NCBI数据库比对,初步将本次调查的蠹虫鉴定为2属3种,分别是双棘长蠹属侧突双棘长蠹(Sinotylon perforans)、双棘长蠹(Sinoxylon anale)和长蠹属槲长蠹(Bostrichus capucinus)。

基于Sanger测序方法检测赛鸽竞翔相关基因的单核苷酸多态性

大量研究发现赛鸽中LDHA、F-KER、DRD4、MSTN和CRY1这5个基因存在单核苷酸多态性,并与归巢能力相关,成为分子辅助育种的潜在SNP标记。当前的检测方法仍以传统的RFLP为主,尚未报道Sanger测序的应用。通过建立一种基于Sanger测序技术的检测方法,以识别这5个基因中的多态性位点。结果表明:针对赛鸽的LDHA、F-KER、DRD4、MSTN和CRY1基因设计了特异性引物,用于扩增单核苷酸多态性位点,所得PCR产物条带单一,显示具有良好的特异性。进一步,利用这些引物对60羽赛鸽的羽毛DNA样品进行扩增和Sanger测序,能成功获得各个基因位点的多态性分型结果。最后,统计了各种基因分型在这60羽赛鸽中的分布频率,发现相关的罕见基因型频率均低于5%,与国外赛鸽群体的频率相一致。研究显示所建立的赛鸽竞翔相关基因SNP分型Sanger测序分析方法,对于选育优良基因型的赛鸽具有重要的应用价值。

Association between childhood obesity and gut microbiota:16S rRNA gene sequencing-based cohort study

BACKGROUND This study aimed to identify characteristic gut genera in obese and normal-weight children(8-12 years old)using 16S rDNA sequencing.The research aimed to provide insights for mechanistic studies and prevention strategies for childhood obesity.Thirty normal-weight and thirty age-and sex-matched obese children were included.Questionnaires and body measurements were collected,and fecal samples underwent 16S rDNA sequencing.Significant differences in body mass index(BMI)and body-fat percentage were observed between the groups.Analysis of gut microbiota diversity revealed lowerα-diversity in obese children.Differences in gut microbiota composition were found between the two groups.Prevotella and Firmicutes were more abundant in the obese group,while Bacteroides and Sanguibacteroides were more prevalent in the control group.AIM To identify the characteristic gut genera in obese and normal-weight children(8-12-year-old)using 16S rDNA sequencing,and provide a basis for subsequent mechanistic studies and prevention strategies for childhood obesity.METHODS Thirty each normal-weight,1:1 matched for age and sex,and obese children,with an obese status from 2020 to 2022,were included in the control and obese groups,respectively.Basic information was collected through questionnaires and body measurements were obtained from both obese and normal-weight children.Fecal samples were collected from both groups and subjected to 16S rDNA sequencing using an Illumina MiSeq sequencing platform for gut microbiota diversity analysis.RESULTS Significant differences in BMI and body-fat percentage were observed between the two groups.The Ace and Chao1 indices were significantly lower in the obese group than those in the control group,whereas differences were not significant in the Shannon and Simpson indices.Kruskal-Wallis tests indicated significant differences in unweighted and weighted UniFrac distances between the gut microbiota of normal-weight and obese children(P<0.01),suggesting substantial disparities in both the species and quantity of gut microbiota between the two groups.Prevotella,Firmicutes,Bacteroides,and Sanguibacteroides were more abundant in the obese and control groups,respectively.Heatmap results demonstrated significant differences in the gut microbiota composition between obese and normal-weight children.CONCLUSION Obese children exhibited lowerα-diversity in their gut microbiota than did the normal-weight children.Significant differences were observed in the composition of gut microbiota between obese and normal-weight children.

Phylogenetic analyses of four species of Ulva and Monostroma grevillei using ITS, rbcL and 18S rDNA sequence data

Chlorophyta species are common in the southern and northern coastal areas of China. In recent years, frequent green tide incidents in Chinese coastal waters have raised concerns and attracted the attention of scientists. In this paper, we sequenced the 18S rDNA genes, the internal transcribed spacer （ITS） regions and the rbcL genes in seven organisms and obtained 536-566 bp long ITS sequences, 1 377-I 407 bp long rbcL sequences and 1 718-1 761 bp long partial 18S rDNA sequences. The GC base pair content was highest in the ITS regions and lowest in the rbcL genes. The sequencing results showed that the three Ulvaprolifera （or U. pertusa） gene sequences from Qingdao and Nan＇ao Island were identical. The ITS, 18S rDNA and rbcL genes in U. prolifera and U. pertusa from different sea areas in China were unchanged by geographic distance. U.flexuosa had the least evolutionary distance from U. californica in both the ITS regions （0.009） and the 18S rDNA （0.002）. These data verified that Ulva and Enteromorpha are not separate genera.

Preliminary study on mitochondrial 16S rRNA gene sequences and phylogeny of flatfishes (Pleuronectiformes)

A 605 bp section of mitochondrial 16S rRNA gene from Paralichthys olivaceus, Pseudorhombus cinnamomeus, Psetta maxima and Kareius bicoloratus, which represent 3 families of Order Pleuronectiformes was amplified by PCR and sequenced to show the molecular systematics of Pleuronectiformes for comparison with related gene sequences of other 6 flatfish downloaded from GenBank. Phylogenetic analysis based on ge- netic distance from related gene sequences of 10 flatfish showed that this method was ideal to explore the rela- tionship between species, genera and families. Phylogenetic trees set-up is based on neighbor-joining, maximum parsimony and maximum likelihood methods that accords to the general rule of Pleuronectiformes evolution. But they also resulted in some confusion. Unlike data from morphological characters, P. olivaceus clustered with K. bicoloratus, but P. cinnamomeus did not cluster with P. olivaceus, which is worth further studying.

Genetic Diversity in Populations of Sepiella maindroni Using 16S rRNA Gene Sequence Analysis

Part of the 16S rRNA gene is amplified with PCR and sequenced for 5 populations of com-mon Chinese cuttlefish Sepiella maindroni: three from the South China Sea, one from East China Sea and one from Japan. The result shows that a total of 5 nucleotide positions are found to have gaps or insertions of base pairs among these individuals, and 13 positions are examined to be variable in all the sequences, which range from 494 to 509 base pairs. All of the individuals are grouped into 7 haplotypes (h1-h7). No marked genetic difference is observed among those populations. All of the individuals from Nagasaki belong to hl and the h3 haplotype is found only in the coastal waters of China. A(?)G transition in Nucleotide 255 is suggested to be taken as a kind of genetic marker to identify the populations distributed in East-South China Sea and the Nagasaki waters of Japan.

Construction of Porphyra yezoensis Pure Line from Protoplasts and Its 18S rDNA Sequence Determination

The wild Porphyra yezoensis collected from the Qingdao coast was used to prepare protoplasts by enzyme digestion. The pure line was constructed by cultivating the protoplasts. The 18S rDNA of the P. yezoensis pure line was cloned and sequenced. Sequence analysis was executed for this sequence and other 22 sequences retrieved from GenBank. A phylogenetic tree was constructed using the neighbor joining method. The results revealed a high diversity of 18S rDNA sequences in genus Porphyra and the considerable variation of 18S rDNA sequences in different strains of the same species P. yezoensis and P. tenera. Significant difference of 18S rDNA sequence was observed between P. yezoensis from Qingdao, China, and the two strains of P. yezoensis from Japan, but the three strains of P. yezoensis formed a stable clade in the phylogenetic tree. These results indicate the possibility of interspecies and intraspecies discrimination of Porphyra using the 18S rDNA sequences.

Genetic relationship between Neobenedenia girellae and N. melleni inferred from 28S rRNA sequences

The fragments of 350 bp in 28S rRNA from the closely related monogenea of trematoda, Neobenedenia girellaeand N. melleni are obtained by polymerase chain reaction (PCR) amplified using a couple of special primers andthen sequenced. The results show that the comparison of 28S rRNA sequences, with only a base varying in 337bp accounting for 0.3% genetic difference, from the relative species N. girellae and N. melleni parasitized on thedifferent fishes in different farms displays that they possess a very high genetic similarity of 99.7%, higher thanthat of 99.41% for the single species N. melleni sampled in different areas, and the intraspecific divergence of N.melleni is 0.59%. Meanwhile, the interspecific differences between the two Neobenedenia and three Benedenia (i.e., B. lutjani, B. rohdei and B. seriolae) range from 2.08% to11.73%. In addition, UPGMA and MP molecularphylogenetic trees are constructed and proved to be consistent with each other. Though the morphologicalcharacteristics and the results of genetic diversity for the two Neobenedenia show a high similarity, whether theybelong to a single species or not are still undefined, and the more genes of them should be further investigated, incombination with the systematical and detailed morphological study.

To Analyze the Sensitivity of RT-PCR Assays Employing S Gene Target Failure with Whole Genome Sequencing Data during Third Wave by SARS-CoV-2 Omicron Variant

Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.

Rice bicoid-related cDNA sequence and its expression during early embryogenesis

Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation. To clone and characterize the rice bicoid-related genes, one cDNA clone, Rb24 (EMBL accession number: AJ2771380), was isolated by screening of rice unmature seed cDNA library. Sequence analysis indicates that Rb24 contains a putative amino acid sequence, which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity, 75% similarity) and involves a lys-9 in putative helix 3. Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner. The transcript was detected strongly in young panicles, but less in young leaves and roots. This results are further confirmed with paraffin section in situ hybridization. The signal is intensive in rice globular embryo and located at the apical tip of the embryo, then, along with the development of embryo, the signal is getting reduced and transfers into both sides of embryo. The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

Strong Law of Large Numbers and Complete Convergence for Sequences of -Mixing Random Variables

We give some theorems of strong law of large numbers and complete convergence for sequences of φ-mixing random variables. In particular, Wittmann＇s strong law of large numbers and Teicher＇s strong law of large nnumbers for independent random variables are generalized to the case of φ -minxing random variables.

Isolation and identification of a sulfate reducing bacteria and sequence analysis of its dissimilatory sulfite reductase gene

A sulfate reducing bacteria was isolated from mining sewage of Daqing Oilfield by Hungate anaerobic technology. Physiological-biochemical analysis showed that the strain could utilize polyacrylamide as sole carbon and nitrogen source. The sequence analysis of 16S rDNA illustrated that the similarity of F8 and Desulfovibrio desulfuricans (AF192153) was 99%, and the similarity sequence of dissimilatory sulfite reductase gene (DSR) cloned from the strain and Desulfovibrio desulfuricans (AF273034) was 98%. Their phylogenitic analysis was basically anastomosed, and thus temporarily named as Desulfovibrio desulfuricans F8. The DSR cloned from F8 strain was 2740 bp in length consisting of three ORF, DSRA, DSRB and DSRD as a single operon (DSRABD) regulated by the same operator. DSRA contained typical conservative box of sulfate—sulfite reducing enzyme (SiteⅠand SiteⅡ), which could bind siroheme and [Fe4S4]. DSRB retained a [Fe4S4] binding site, with an uncomplimentary structure for siroheme binding. There was no conservative box in DSRD. Sequence analysis of DSR will provide a theoretical basis for quantitative detection, metabolic pathway modification through gene engineering, and sulfate reducing bacteria (SRB) suppression.

Spatiotemporal evolution of the 2021 M_(S)6.4 Yangbi earthquake sequence

Based on the seismic phase reports of the Yangbi area from January 1 to June 25,2021,and the waveform data of M≥4 earthquakes,we obtained the relocation results and focal mechanism solutions of the M_(S)6.4 Yangbi earthquake sequence using the HypoDD and CAP methods.Based on our results,our main conclusions are as follows:(1)the M_(S)6.4 Yangbi earthquake sequence is a typical foreshock-mainshock-aftershock sequence.The fore-shocks of the first two stages have the obvious fronts of migration and their migration rate increased gradually.There was no apparent front of migration during the third stage,and the occurrence of the mainshock was related to stress triggering from a M5.3 foreshock.We tentatively speculate that the rupture pattern of the Yangbi earthquake sequence conforms to the cascading-rupture model;and(2)the main fault of the M_(S)6.4 Yangbi earthquake sequence is a NW-trending right-lateral strike-slip fault.As time progressed,a minor conjugate aftershock belt formed at the northwest end of this fault,and a dendritic branching structure emerged in the southern fault segment,showing a complex seismogenic fault structure.We suggested that the fault of the Yangbi earthquake sequence may be a young sub-fault of the Weixi-Weishan fault.

The modulating of Qingguang’anⅡFormula on gut microbiota in mice with chronic high intraocular pressure by 16S rDNA sequencing

Objective To investigate the effects of Qingguang'anⅡFormula(QGAⅡ)on the gut micro-biota of mice with chronic high intraocular pressure(IOP)model,and explore its key micro-biota for protecting the optic nerve.Methods A total of 10 specific pathogen free(SPF)grade female DBA/2J mice were random-ly divided into model group and QGAⅡgroup(n=5 for each group),while additional 5 SPF-grade female C57BL/6J mice were assigned to control group.Mice presented spontaneous high IOP and showed elevated approximately at the age of seven months.The high IOP was maintained until week 38,when gavage was initiated.Mice in control group underwent the same intragastric treatment,while those in QGAⅡgroup were gavaged with QGAⅡ(9.67 g/kg),once a day for four weeks.Retinal morphology was examined using hematoxylin and eosin(HE)staining,with the number of retinal ganglion cells(RGCs)counted.The expression level of Brn3a protein,a specific marker for RGCs,was detected by immunofluorescence,with the mean optical density(OD)measured for quantitative analysis.In addition,16S rDNA se-quencing was leveraged to analyze changes in the diversity of gut microbiota,including theirα-diversity(Chao1,Shannon,Pielou’s evenness,and observed species index)andβ-diversity.Venn diagrams and linear discriminant analysis effect size(LEfSe)analysis was employed to investigate the number of amplicon sequence variants(ASVs),the abundance of differential gut microbiota species,and the classification of species at both the phylum and genus levels within the three groups of mice.Results HE staining revealed that compared with control group,model group showed signif-icant reduction in the number of RGCs(P<0.01),with intracellular vacuolar degeneration and nuclear pyknosis.After QGAⅡtreatment,the number of RGCs was significantly in-creased compared with model group(P<0.01),with notable improvements in intracellular vacuolar degeneration.Immunofluorescence analysis showed that the mean OD of Brn3a protein was significantly decreased in model group compared with control group(P<0.01),while QGAⅡtreatment significantly elevated its expression level(P<0.01).Analysis ofα-diversity showed that after QGAⅡintervention,the Chao1,Shannon,and Pielou’s evenness indices were significantly increased(P<0.01),and the observed species index was elevated(P<0.05).β-Diversity analysis demonstrated distinct clustering among the three groups,indicating relatively low similarity in bacterial community structures.ASV clustering identi-fied a total of 14061 ASVs across all groups,with 9514 ASVs shared between model and QGAⅡgroups.At the phylum level,the abundance of Bacteroidetes was significantly decreased in model group compared with control group(P<0.01),while Firmicutes and the Firmicutes/Bacteroidetes(F/B)ratio were significantly increased(P<0.01).QGAⅡtreatment significantly reduced both Firmicutes abundance and the F/B ratio(P<0.01).At the genus level,Lactobacillus was dominant across all groups,with its abundance significantly in-creased in model group(P<0.01)and subsequently decreased following QGAⅡintervention(P<0.05).Conclusion QGAⅡrestructured the gut microbiota of DBA/2J mice with chronic high IOP,bringing changes in their diversity and abundance of components.Firmicutes,Bacteroidetes,Lactobacillus,along with their associated microorganisms,are likely critical components of the gut microbiota that contribute to the optic neuroprotective effects of QGAⅡon chronic high IOP mice.

Tracking a maneuvering target in clutter with out-of-sequence measurements for airborne radar

There are many proposed optimal or suboptimal al- gorithms to update out-of-sequence measurement（s） （OoSM（s）） for linear-Gaussian systems, but few algorithms are dedicated to track a maneuvering target in clutter by using OoSMs. In order to address the nonlinear OoSMs obtained by the airborne radar located on a moving platform from a maneuvering target in clut- ter, an interacting multiple model probabilistic data association （IMMPDA） algorithm with the OoSM is developed. To be practical, the algorithm is based on the Earth-centered Earth-fixed （ECEF） coordinate system where it considers the effect of the platform＇s attitude and the curvature of the Earth. The proposed method is validated through the Monte Carlo test compared with the perfor- mance of the standard IMMPDA algorithm ignoring the OoSM, and the conclusions show that using the OoSM can improve the track- ing performance, and the shorter the lag step is, the greater degree the performance is improved, but when the lag step is large, the performance is not improved any more by using the OoSM, which can provide some references for engineering application.

Rearrangement Inequality and Chebyshev＇s Sum Inequality on Positive Tensor Products of Orlicz Sequence Space with Banach Lattice

Let φ be an Orlicz function that has a complementary function φ＊ and let lφ be an Orlicz sequence space. We prove a similar version of Rearrangement Inequality and Chebyshev＇s Sum Inequality in lφ FX, the Fremlin projective tensor product of lφ with a Banach lattice X, and in lφ iX, the Wittstock injective tensor product of lφ with a Banach lattice X.