Analysis of the chloroplast genome of Incarvillea younghusbandii Sprague / 生物工程学报

Incarvillea younghusbandii Sprague is a traditional tonic herb. The roots are used as herbal medicine for nourishing and strengthening, as well as treating postpartum milk deficiency and weakness. In this study, the chloroplast genome of I. younghusbandii was sequenced and assembled by the high-throughput sequencing technology. The sequence characteristics, sequence repeats, codon usage bias, phylogenetic relationships and estimated divergence time of I. younghusbandii were analyzed. The 159 323 bp sequence contained a large single copy (80 197 bp), a small single copy (9 030 bp) and two inverted repeat sequences (35 048 bp). It contained 120 genes, including 77 protein coding genes, 8 ribosomal RNA genes and 35 transfer RNA genes. AAA was the most frequent codon in the chloroplast coding sequence of I. younghusbandii. A total of 42 simple sequence repeats were identified in the chloroplast genome. Phylogenetic analysis revealed I. younghusbandii was mostly like its taxonomically close relative Incarvillea compacta. The divergence between I. younghusbandii and I. compacta was dated to 4.66 million years ago. This study was significant for the scientific conservation and development of resources related to I. compacta. It also provides a basic genetic resource for the subsequent species identification of the genus Incarvillea, and the population genetic diversity study of Bignoniaceae.

Whole genome sequence analysis of the first reported isolate of Salmonella Agona carrying blaCTX-M-55 gene in Brazil

This study analyzes the genomic findings of the first report of Salmonella isolate carrying the blaCTX-M-55 gene, recovered from a bacteremic patient from Brazil. A bacterial isolate positive for the blaCTX-M-55 gene was submitted to antimicrobial susceptibility testing by disk diffusion and epsilometric test. Whole genome sequencing was performed using Illumina technology. Conjugation assay was performed; plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinION long reads sequencing. Isolate 288_18 was identified as sequence type ST13, resistant to ampicillin, cefotaxime, ceftazidime, cefepime, ceftriaxone, and aztreonam. A transferable IncFII plasmid sized approximately 67 kb was found to carry the blaTEM-1 and blaCTX-M-55 in a module consisting of IS26-blaTEM-1B-WbuC-blaCTX-M-55-IS26. In addition, an 117 kb IncI1plasmid was also identified in the 288_18 isolate, but without additional resistance genes. To the best of our knowledge, this is the first report of blaCTX-M-55 in Salmonella isolated from human infection in Brazil. The occurrence of blaCTX-M-55 in the IncFII epidemic plasmid in a relevant clinical human isolate of Salmonella Agona underscores the urgent need for enhanced and effective continuous surveillance for controlling its dissemination.

Complete chloroplast genome sequencing and phylogeny of wild Atractylodes lancea from Yuexi, Anhui province / 中国中药杂志

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.

Marcadores farmacogenéticos baseados em dados genômicos de uma coorte de idosos da cidade de São Paulo e o panorama da farmacogenômica no Brasil / Pharmacogenetic markers based on genomic data from a cohort of elderly individuals from the city of São Paulo and the landscape of pharmacogenomics research in Brazil

A medicina de precisão trata-se de uma área tem avançado rapidamente nos últimos anos, juntamente com o desenvolvimento de novas tecnologias de diagnóstico e tratamento, levando à minimização de efeitos colaterais relacionados ao tratamento melhoras nos resultados clínicos de forma geral. A pesquisa em farmacogenômica (PGx) desempenha um importante papel na área da medicina de precisão, ao investigar as variantes genéticas que modulam a resposta a fármacos, por meio de alterações em sua farmacocinética (PK) ou farmacodinâmica (PD). A distribuição de variantes de farmacogenes difere consideravelmente entre as populações e o sequenciamento genético completo em populações diversas (WGS, do inglês whole-genome sequencing) desempenha um papel importante como uma abordagem de sequenciamento abrangente para detectar variantes comuns e raras. Objetivo: Os objetivos gerais foram: i) verificar o panorama dos estudos brasileiros na área da PGx, em termos de metodologia de estudo e oportunidades potenciais na área de pesquisa no Brasil, e ii) avaliar a frequência de marcadores farmacogenéticos em idosos da cidade de São Paulo e avaliar a proporção de indivíduos potencialmente em alto risco de interações farmacogenéticas no ano de 2010. Métodos: Na primeira parte do trabalho, foi realizada uma revisão sistemática que buscou estudos brasileiros na área da PGx que analisaram a associação entre fármaco(s) e gene(s) que apresentam interesse especial na área da farmacogenética (VIPs, do inglês Very Important Pharmacogenes); foram incluídos 97 estudos para análise do texto completo. Na segunda parte do trabalho, a ferramenta Stargazer foi utilizada para identificar star alleles de 38 farmacogenes, utilizando o banco de dados de WGS de 1.171 indivíduos não-relacionado do estudo SABE (Estudo de Saúde, Bem-Estar e Envelhecimento). Resultados: Na revisão da literatura, 32 dos 65 VIPs foram analisados por estudos de associação na população brasileira. Noventa e seis eram estudos de genes candidatos e um era GWAS (do inglês, genome-wide association study). Agentes antitrombóticos, fármacos que atuam no sistema nervoso e no sistema cardiovascular foram as classes mais estudadas. Em geral, 68% eram estudos observacionais e 24% eram ensaios clínicos. A análise dos marcadores farmacogenéticos na coorte SABE mostrou que 352 haplótipos ou star alleles únicos foram observados em todos os 38 farmacogenes avaliados. Destes, 255 apresentaram frequência < 0,05 e 199 apresentaram frequência < 0,01; 70,1% dos star alleles foram classificados como tendo perda ou diminuição de função, ou função desconhecida. Para os onze farmacogenes com alto nível de evidência de interação com medicamentos (1A) segundo o Consórcio de Implementação de Farmacogenética Clínica (CPIC, do inglês Clinical Pharmacogenetics Implementation Consortium), verificou-se que mais de 99% dos indivíduos carregavam pelo menos um genótipo de alto risco. Segundo o registro de medicamentos da coorte, 22,5% dos indivíduos que utilizavam um medicamento com nível de evidência do 1A (CPIC) estavam potencialmente em risco de uma interação farmacogenética por possuírem um fenótipo predito que interage com o medicamento em uso. Conclusão: Populações miscigenadas ainda estão sub-representadas em grandes estudos genômicos, e este projeto pode contribuir com dados adicionais de para PGx na população brasileira.

The area of precision medicine is growing rapidly with advances in diagnostic and treatment options, leading to improvements in clinical outcomes and minimization of unnecessary side effects. Pharmacogenomics (PGx) plays an important role in precision medicine and deals with the variation of drug response due to genetic factors. Research in the field of PGx aims to identify genetic variants that modulate the response to drugs, through alterations in their pharmacokinetics (PK) or pharmacodynamics (PD). The distribution of PGx variants differs considerably among populations, and whole-genome sequencing (WGS) performed on diverse populations plays a major role as a comprehensive sequencing approach to detect both common and rare variants. Objective: This study evaluated i) the landscape of Brazilian PGx studies in terms of study methodology and potential opportunities in this research area in Brazil, and ii) the frequency PGx markers in a cohort of elderly individuals from the city of São Paulo, and the proportion of individuals at a potential high-risk for PGx interaction in 2010. Methods: In the first part of this research, a systematic review was performed to find Brazilian studies in the area of PGx which analyzed the association between drugs and very important pharmacogenes (VIP); 97 studies were included for full-texts review. In the second part, the tool Stargazer was used to call star alleles from 38 pharmacogenes using data from the Health, Well-Being, and Aging Study (SABE), which includes variants from whole-genome sequences of 1,171 unrelated individuals. Results: In the literature review, 32 out of 65 VIPs were analyzed by association studies in the Brazilian population. Ninety-six were candidate gene studies and one was GWAS. Antithrombotic agents, drugs that act on the nervous system, and the cardiovascular system were the most studied dugs. In general, 68% comprised observational studies and interventional clinical trials accounted for 24%. The analysis of PGx markers in the SABE cohort showed 352 unique star alleles or haplotypes in all 38 pharmacogenes assessed. Among these, 255 and 199 had a frequency < 0.05 and < 0.01, respectively, with decreased/ loss-of-function/ unknown function corresponding to 70.1%. For eleven pharmacogenes with high level of evidence (1A) supporting the association with drugs according to CPIC (Clinical Pharmacogenetics Implementation Consortium), more than 99% of the individuals carried at least one high risk genotype. According to cohort medication register from 2010 round of data collection, 22.5% of individuals using CPIC level 1A drugs were potentially at high risk for a pharmacogenetic interaction because they also had a predicted phenotype which interacts with the drug taken. Conclusion: Admixed populations are still underrepresented in large genomic studies, and this project could provide insights to PGx in those populations.

Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

Application of the artificial intelligence-rapid whole-genome sequencing diagnostic system in the neonatal/pediatric intensive care unit / 中国当代儿科杂志

Pediatric patients in the neonatal intensive care unit (NICU) and the pediatric intensive care unit (PICU) have a high incidence rate of genetic diseases, and early rapid etiological diagnosis and targeted interventions can help to reduce mortality or improve prognosis. Whole-genome sequencing covers more comprehensive information including point mutation, copy number, and structural and rearrangement variations in the intron region and has become one of the powerful diagnostic tools for genetic diseases. Sequencing data require highly professional judgment and interpretation and are returned for clinical application after several weeks, which cannot meet the need for the diagnosis and treatment of genetic diseases in children. This article introduces the clinical application of rapid whole-genome sequencing in the NICU/PICU and briefly describes related techniques of artificial intelligence-rapid whole-genome sequencing diagnostic system, a rapid high-throughput automated platform for the diagnosis of genetic diseases. The diagnostic system introduces artificial intelligence into the processing of data after whole-genome sequencing and can solve the problems of long time and professional interpretation required for routine genome sequencing and provide a rapid diagnostic regimen for critically ill children suspected of genetic diseases within 24 hours, and therefore, it holds promise for clinical application.

Genome-wide analysis of aberrant DNA methylation patterns in iPSCs derived from patients with Down syndrome / 中华医学遗传学杂志

OBJECTIVE@#To study the correlation between DNA methylation patterns and gene expression in Down syndrome (DS).@*METHODS@#Induced pluripotent stem cells (iPSCs) derived from normal controls and DS patients were subjected to whole genome bisulfite sequencing and differentially methylated region (DMR) screening. Statistical analysis for chromosomal and gene element distribution were carried out for DMR. Gene ontology (GO) and enrichment-based cluster analysis were used to explore the molecular function of differentially expressed genes.@*RESULTS@#A total of 1569 DMR were identified in iPSCs derived from DS patients, for which the proportion of hypermethylation in promoter regions was significantly greater than that of the genebody. No DMR enrichment was noted on chromosome 21. Hypermethylation of the promoter and genebody was predicted to be inhibitory for gene expression. Functional clustering revealed the pathways related to neurodevelopmental, stem cell pluripotency and organ size regulation to be significantly correlated with differentially methylated genes.@*CONCLUSION@#Extensive and stochastic anomalies of genome-wide DNA methylation has been discovered in iPSCs derived from DS patients, for which the pattern and molecular regulation of methylation were significantly different from those of normal controls. Above findings suggested that DNA methylation pattern may play a vital role in both the pathogenesis of neurodevelopmental disorders and other phenotypic abnormalities during early embryonic development.

Evaluation of Multidrug Resistant Loop-mediated Isothermal Amplification Assay for Detecting the Drug Resistance of / 生物医学与环境科学（英文）

Objective@#To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the @*Methods@#MDR-LAMP assay was evaluated using 100 @*Results@#The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of @*Conclusion@#MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of

Immunogenicity of Whole / 生物医学与环境科学（英文）

Objectives@#To evaluate the immunogenicity of @*Methods@#Protein extracts from @*Results@#Immunization with @*Conclusion@#This is the advanced study to investigate the immunogenicity of

Complete genome sequencing of Pseudomonas syringae pv. actinidiae Biovar 3, P155, kiwifruit pathogen originating from China / Sequenciamento completo do genoma Pseudomonas syringae pv. actinidiae Biovar 3, P155, agente patogênico do kiwi, originário da China

Pseudomonas syringae pv. actinidiae is a bacterial pathogen of kiwifruit. Based on the results of the pathogenicity assay, we sequenced the strain Pseudomonas syringae (Psa3) P155 which possesses a series of virulence and resistance genes, CRISPR candidate elements, prophage related sequences, methylation modifications, genomic islands as well as one plasmid. Most importantly, the copper resistance genes copA, copB, copC, copD, and copZ as well as aminoglycoside resistance gene ksgA were identified in strain P155, which would pose a threat to kiwifruit production. The complete sequence we reported here will provide valuable information for a better understanding of the genetic structure and pathogenic characteristics of the genome of P155.

Pseudomonas syringae pv. actinidiae agente causal do cancro bacteriano do kiwi. Com base nos resultados do teste de patogenicidade, foi sequenciado um isolado de Pseudomonas syringae (Psa3) P155, que abriga a uma série de genes de virulência e resistência, elementos candidatos CRISPR, sequências relacionadas a profagos, modificações na metilação, ilhas genômicas, e também um plasmídeo. O mais importante foram os genes de resistência ao cobre, copA, copB, copC, copD e copZ, bem como, o gene de resistência aminoglicosídea ksgA identificados na estirpe P155, os quais representariam uma ameaça à produção de kiwi. A sequência completa relatada fornecerá informações valiosas para uma melhor compreensão da estrutura genética e as características patogênicas do genoma de P155.

Citogenética de las hemopatías malignas en la era de la secuenciación / Cytogenetics of malignant hemopathies in the sequencing era

Las neoplasias hematológicas se caracterizan por un gran número y complejidad de alteraciones genéticas, desde la formación de genes de fusión a partir de translocaciones e inversiones cromosómicas hasta mutaciones génicas y alteraciones epigenéticas que han permitido la identificación de nuevos oncogenes y genes supresores de tumores responsables de su etiología. Al abordar el estudio genético de las leucemias se utilizan múltiples técnicas como la citogenética convencional, citogenética molecular (hibridaciónin situ por fluorescencia (FISH), esta última con una mayor sensibilidad, especificidad y rapidez que permiten el diagnóstico, la estratificación pronóstica y seguimiento de la enfermedad. Las técnicas anteriores se integran con técnicas de biología molecular, secuenciación génica, entre otras, que permiten el hallazgo de nuevos marcadores genéticos con una mejor caracterización de las hemopatías malignas y la posibilidad del desarrollo de nuevos fármacos específicos que actúen sobre la diana molecular. El objetivo fue revisar la utilidad de la citogenética y la secuenciación génica en el estudio de la leucemia mieloide aguda y la leucemia linfocítica crónica. Ante las ventajas, desventajas y limitaciones de estas técnicas genéticas es necesario utilizarlas de forma complementaria y nunca excluyente(AU)

Hematological neoplasms are characterized by a large number and great complexity of genetic disorders, from the formation of fusion genes after chromosomal translocations and inversions to gene mutation and epigenetic disorders that have permitted the identification of new oncogenes and tumor-suppressing genes responsible for their etiology. When addressing the genetic study of leukemias, multiple techniques are used, such as conventional cytogenetics, molecular cytogenetics, and fluorescence in situ hybridization (FISH), the latter having the higher degree of sensitivity, specificity and speed, which allow diagnosis, prognostic stratification and follow-up of the disease. The previous techniques are integrated with molecular biology techniques, gene sequencing, among others, which allow discovery of new genetic markers with better characterization of malignant hemopathies and the possibility of developing new specific drugs against the molecular target. The objective was to review the usefulness of cytogenetics and gene sequencing in the study of acute myeloid leukemia and chronic lymphocytic leukemia. Given the advantages, disadvantages and limitations of these genetic techniques, it is necessary to use them in as complementary but never exclusive management ways(AU)

Diversidad molecular de variantes patogénicas de Vibrio parahaemolyticus en el Perú / Molecular diversity in pathogenic variants of vibrio parahaemolyticus in Peru

RESUMEN Con el objetivo de determinar la diversidad de variantes patogénicas de Vibrio parahaemolyticus en el Perú durante el periodo 1995-2017, se analizaron 102 genomas peruanos (97 clínicos y 5 ambientales) empleando el esquema de tipificación multilocus y BLASTn para la búsqueda de genes de virulencia. Se identificaron 15 tipos de secuencia diferentes, encontrándose que el genotipo ST3, perteneciente al clon pandémico, fue el más abundante, con 52% (n=53); seguido por el ST120, con 23,5% (n=24); y el complejo clonal CC345, con 11,8% (n=12). Un total de 89 cepas analizadas presentaron genes que codifican la isla de patogenicidad VpaI-7 (87,3%), mientras que 96 presentaron el gen tdh (94,1%), y 6, el trh (5,9%). Durante el periodo evaluado, se resalta la predominancia del ST3, causante de un importante brote en el pasado del Perú, además de otros genotipos patógenos que representan un riesgo latente en salud pública asociado al consumo de alimentos marinos.

ABSTRACT During the period from 1995 to 2017, in order to determine the diversity of Vibrio parahaemolyticus pathogenic variants in Peru, 102 Peruvian genomes (97 from a hospital setting and 5 from an out-of-hospital setting) were analyzed using the multilocus typification scheme and BLASTn in the search for virulence genes. Fifteen different sequence types were identified. It was found that the ST3 genotype, which is found in the pandemic clone, was the most abundant, with 52% (n=53); followed by ST120, with 23.5% (n=24); and the CC345 clonal complex, with 11.8% (n=12). A total of 89 analyzed strains presented genes encoding the pathogenicity island VpaI-7 (87.3%), while 96 presented the tdh gene (94.1%), and 6 the trh gene (5.9%). The ST3 genotype was the predominant one during the evaluated period, this genotype was the cause of a major outbreak in Peru's past history. Other pathogenic genotypes found represent a latent public health risk associated with seafood consumption.

Evaluation of drug susceptibility profile of Mycobacterium tuberculosis Lineage 1 from Brazil based on whole genome sequencing and phenotypic methods

BACKGROUND The evaluation of procedures for drug susceptibility prediction of Mycobacterium tuberculosis based on genomic data against the conventional reference method test based on culture is realistic considering the scenario of growing number of tools proposals based on whole-genome sequences (WGS). OBJECTIVES This study aimed to evaluate drug susceptibility testing (DST) outcome based on WGS tools and the phenotypic methods performed on isolates of M. tuberculosis Lineage 1 from the state of Pará, Brazil, generally associated with low levels of drug resistance. METHODOLOGY Culture based DST was performed using the Proportion Method in Löwenstein-Jensen medium on 71 isolates that had been submitted to WGS. We analysed the seven main genome sequence-based tools for resistance and lineage prediction applied to M. tuberculosis and for comparison evaluation we have used the Kappa concordance test. FINDINGS When comparing the WGS-based tools against the DST, we observed the highest level of agreement using TB-profiler. Among the tools, TB-profiler, KvarQ and Mykrobe were those which identified the largest number of TB-MDR cases. Comparing the four most sensitive tools regarding resistance prediction, agreement was observed for 43 genomes. MAIN CONCLUSIONS Drug resistance profiling using next-generation sequencing offers rapid assessment of resistance-associated mutations, therefore facilitating rapid access to effective treatment.

Development of nuclear SSR and chloroplast genome markers in diverse Liriodendron chinense germplasm based on low-coverage whole genome sequencing

BACKGROUND: Liriodendron chinense ranges widely in subtropical China and northern Vietnam; however, it inhabits several small, isolated populations and is now an endangered species due to its limited seed production. The objective of this study was to develop a set of nuclear SSR (simple sequence repeats) and multiple chloroplast genome markers for genetic studies in L. chinense and their characterization in diverse germplasm. RESULTS: We performed low-coverage whole genome sequencing of the L. chinense from four genotypes, assembled the chloroplast genome and identified nuclear SSR loci by searching in contigs for SSR motifs. Comparative analysis of the four chloroplast genomes of L. chinense revealed 45 SNPs, 17 indels, 49 polymorphic SSR loci, and five small inversions. Most chloroplast intraspecific polymorphisms were located in the interspaces of single-copy regions. In total, 6147 SSR markers were isolated from low-coverage whole genome sequences. The most common SSR motifs were dinucleotide (70.09%), followed by trinucleotide motifs (23.10%). The motif AG/TC (33.51%) was the most abundant, followed by TC/AG (25.53%). A set of 13 SSR primer combinations were tested for amplification and their ability to detect polymorphisms in a set of 109 L. chinense individuals, representing distinct varieties or germplasm. The number of alleles per locus ranged from 8 to 28 with an average of 21 alleles. The expected heterozygosity (He) varied from 0.19 to 0.93 and the observed heterozygosity (Ho) ranged from 0.11 to 0.79. CONCLUSIONS: The genetic resources characterized and tested in this study provide a valuable tool to detect polymorphisms in L. chinense for future genetic studies and breeding programs.

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis / 基因组蛋白质组与生物信息学报·英文版

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.

Improving crops genome through genetic engineering of the key metabolic pathways

Soybean loss due to pests and pathogens is a serious problem worldwide. Soybean producers have few options to manage diseases caused by general pathogens where major genes for full resistance have not been discovered. The innate defense of soybean plants could be enhanced by improving content and composition of lignin by genetic engineering of the phenylpropanoid pathway.We used a novel technique of germ-line genetic transformation of soybean plants via natural pollen tubes as vectors. This technique uses Agrobacterium tumefaciensto mediate transfer of genes of interest to the zygote to introduce the key lignification genes (PtMYB4, PAL5, F5H, CAD1) into soybean genome. We observed 5.6% average transformation efficiency in the first generation of transgenic plants and in the second generation the presence of the transgene constructs was confirmed in more than 50% (for CsVMV/PtMYB4sens, 35SVTM/PAL5, C4H/F5H, CsVMV/CAD1constructs) transgenic soybean lines. We confirmed the expression of the introduced genes at transcriptional level using RT-PCR and Northern blot. Functional analysis using lignin content determination and the activity of PAL5 and CAD1 enzymes demonstrated that the transgenes perform their function in planta. The proposed technique is effectiveand inexpensive and can be used to create novel stress and disease resistant soybean genotypes.

Use of uracil-DNA glycosylase enzyme to reduce DNA-related artifacts from formalin-fixed and paraffin-embedded tissues in diagnostic routine

Background: Detection of somatic mutations is a mandatory practice for therapeutic definition in precision oncology. However, somatic mutation detection protocols use DNA from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, which can result in detection of nonreproducible sequence artifacts, especially C:G > T:A transitions, in DNA. In recent studies, DNA pretreatment with uracil DNA glycosylase (UDG), an enzyme involved in base excision repair, significantly reduced the number of DNA artifacts after mutation detection by next-generation sequencing (NGS) and other methods, without affecting the capacity to detect real mutations. This study aimed to evaluate the effects of UDG enzymatic pretreatment in reducing the number of DNA sequencing artifacts from FFPE tumor samples, to improve the accuracy of genetic testing in the molecular diagnostic routine. Methods: We selected 12 FFPE tumor samples (10 melanoma, 1 lung, and 1 colorectal tumor sample) with different storage times. We compared sequencing results of a 16-hotspot gene panel of NGS libraries prepared with UDG-treated and untreated samples. Results: All UDG-treated samples showed large reductions in the total number of transitions (medium reduction of 80%) and the transition/transversion ratio (medium reduction of 75%). In addition, most sequence artifacts presented a low variant allele frequency (VAF < 10%) which are eliminated with UDG treatment. Conclusion: Including UDG enzymatic treatment before multiplex amplification in the NGS workflow significantly decreased the number of artifactual variants detected in FFPE samples. Thus, including this additional step in the current methodology should improve the rate of true mutation detection in the molecular diagnostic routine.

Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates / 基因组蛋白质组与生物信息学报·英文版

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the 11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years. Species and drug specific resistance patterns were observed including increased resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species, including conserved essential genes and known resistance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene-drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge at https://gear-base.com.

Application of whole genome sequencing technology and bioinformatics analysis in antimicrobial resistance researches / 生物工程学报

The emergence and spread of antimicrobial resistance has become a serious global issue. Bacterial characteristics, such as antimicrobial resistance genes, virulence-associated genes, plasmid types, and phylogenetic relationship among different strains, are the keys to unravel the occurrence and dissemination of antimicrobial resistance. However, the accuracy and efficiency of the traditional techniques, such as polymerase chain reaction and pulsed field gel electrophoresis is insufficient to underlying the mystery of antimicrobial resistance. Recently, the whole genome sequencing and high-throughput bioinformatics analysis have been successfully used in antimicrobial resistance studies, helping scientists to obtain the nature of antimicrobial resistance bacteria quickly, and more precisely to paint the evolutionary relationship among different strains. Therefore, in this study, we aim to systematically introduce the recent development of whole genome sequencing analysis, including different methods and corresponding characteristics of library preparation, platform sequencing, data analysis, and the latest application of the technology in the antimicrobial resistance research. We hope that this review can provide more comprehensive knowledge about whole genome sequencing and bioinformatic analysis for antimicrobial resistance research.

Practical stability of whole-genome bisulfite sequencing using plasma cell-free DNA / 生物工程学报

With the development of liquid biopsy technology, plasma cell-free DNA (cfDNA) becomes one of the research hotspots. Whole-genome bisulfite sequencing of plasma cell-free DNA has shown great potential medical applications such as cancer detection. However, the practical stability evaluation is still lacking. In this study, plasma cell-free DNA samples from two volunteers at different time were collected and prepared for sequencing in multiple laboratories. The library preparation strategies include pre-bisulfite, post-bisulfite and regular whole-genome sequencing. We established a set of quality control references for plasma cell-free DNA sequencing data and evaluated practical stability of blood collection, DNA extraction, and library preparation and sequencing depth. This work provided a technical practice guide for the application of plasma cfDNA methylation sequencing for clinical applications.