Single molecular real-time(SMRT)sequencing,also called third-generation sequencing,is a novel sequencing technique capable of generating extremely long contiguous sequence reads.While conventional short-read sequencin...Single molecular real-time(SMRT)sequencing,also called third-generation sequencing,is a novel sequencing technique capable of generating extremely long contiguous sequence reads.While conventional short-read sequencing cannot evaluate the linkage of nucleotide substitutions distant from one another,SMRT sequencing can directly demonstrate linkage of nucleotide changes over a span of more than 20 kbp,and thus can be applied to directly examine the haplotypes of viruses or bacteria whose genome structures are changing in real time.In addition,an error correction method(circular consensus sequencing)has been established and repeated sequencing of a single-molecule DNA template can result in extremely high accuracy.The advantages of long read sequencing enable accurate determination of the haplotypes of individual viral clones.SMRT sequencing has been applied in various studies of viral genomes including determination of the full-length contiguous genome sequence of hepatitis C virus(HCV),targeted deep sequencing of the HCV NS5A gene,and assessment of heterogeneity among viral populations.Recently,the emergence of multi-drug resistant HCV viruses has become a significant clinical issue and has been also demonstrated using SMRT sequencing.In this review,we introduce the novel third-generation PacBio RSII/Sequel systems,compare them with conventional next-generation sequencers,and summarize previous studies in which SMRT sequencing technology has been applied for HCV genome analysis.We also refer to another long-read sequencing platform,nanopore sequencing technology,and discuss the advantages,limitations and future perspectives in using these thirdgeneration sequencers for HCV genome analysis.展开更多
Global concerns have been paid to the potential hazard of traditional herbal medicinal products(THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplemen...Global concerns have been paid to the potential hazard of traditional herbal medicinal products(THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, realtime(SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan(JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing(CCS) reads belonging to the ITS2 and psb A-trn H regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.展开更多
酒药是传统黄酒酿造过程中重要的动力源,具有糖化和发酵的双重作用,绍兴酒药赋予绍兴酒独特的品质。为探究绍兴地区不同酒厂酒药微生物组成、理化指标及其特性,首先通过单分子实时定量测序技术(single molecule real time sequencing,SM...酒药是传统黄酒酿造过程中重要的动力源,具有糖化和发酵的双重作用,绍兴酒药赋予绍兴酒独特的品质。为探究绍兴地区不同酒厂酒药微生物组成、理化指标及其特性,首先通过单分子实时定量测序技术(single molecule real time sequencing,SMRT-seq)研究酒药的微生物组成,然后测定酒药水分含量、酸度及酶活性等相关理化指标,最后筛选鉴定酒药中核心微生物并研究其功能特性。微生物群落结构分析结果表明,不同酒厂酒药细菌群落种类及相对丰度差异较大,以戊糖片球菌(Pediococcus pentosaceus)、食窦魏斯氏菌(Weissella cibaria)、肠杆菌属为主(Enterococcus);真菌较为稳定,以扣囊复膜酵母(Saccharomycopsis fibuligera)为主。理化指标结果显示,JH酒厂酒药水分含量(14.52%)显著高于其他酒厂样品(P<0.05),TP和JH酒厂的酒药酸度(33.14和35.49 g/kg)和液化力(1.4和1.31 U/g)显著高于其他酒厂样品(P<0.05),SYH酒厂酒药的酸性蛋白酶活力最高,为190.08 U/g(P<0.05)。不同厂区酒药存在一定差异,但在核心功能性微生物组成及特性方面具有一致性,筛选得到71株微生物,功能分析结果显示扣囊复膜酵母对酒药酵母的淀粉酶和蛋白酶活性贡献最大,异常威克汉姆酵母可能是潜在的风味贡献者。该研究为未来酒药机械化生产提供理论依据,筛选获得的功能微生物能够为机械化黄酒风味提升提供菌种。展开更多
Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. ~To better understand the role of the surface-associated l...Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. ~To better understand the role of the surface-associated lifestyle of C. marina and the phylogeny of the family Halomonadaceae, we sequenced the entire genome of C. marina JCM 21022 ~T using single molecule real-time sequencing technology(SMR^T) and performed comparative genomics and phylogenomics analyses. ~The circular chromosome was 4 176 300 bp with an average GC content of 62.44% and contained 3 611 predicted coding sequences, 72 t RNA genes, and 21 r RNA genes. ~The C. marina JCM 21022 ~T genome contained a set of crucial genes involved in surface colonization processes. ~The comparative genome analysis indicated the significant diff erences between C. marina JCM 21022 ~T and Cobetia amphilecti KMM 296(formerly named C. marina KMM 296) resulted from sequence insertions or deletions and chromosomal recombination. Despite these diff erences, pan and core genome analysis showed similar gene functions between the two strains. ~The phylogenomic study of the family Halomonadaceae is reported here for the first time. We found that the relationships were well resolved among every genera tested, including Chromohalobacter, Halomonas, Cobetia, Kushneria, Zymobacter, and Halotalea.展开更多
Intestinal dysbiosis is closely related to a variety of medical conditions,especially gastrointestinal diseases.The present study aimed to investigate the effects of koumiss on chronic atrophic gastritis(CAG)in an out...Intestinal dysbiosis is closely related to a variety of medical conditions,especially gastrointestinal diseases.The present study aimed to investigate the effects of koumiss on chronic atrophic gastritis(CAG)in an outpatient clinical trial(n=10;all female subjects aged 41-55;body mass index ranging from 19.5 to 25.8).Each patient consumed three servings of koumiss per day(i.e.250 ml daily before each of 3 meals)for a 60-day period.The improvement of patients'symptoms was monitored by comparing the total scores of symptoms before and after the treatment.Meanwhile,the changes in the patients’fecal microbiota composition and specific blood parameters were determined.After the 60-day koumiss administration,significant symptom improvements were observed,as evidenced by the reduction of the total symptoms score,and changes in blood platelet and cholesterol levels.The changes in patients’fecal microbiota composition were found.The patients’fecal microbiota fell into two distinct enterotypes,Bacteroides dorei/Bacteroides uniformis(BB-enterotype)and Prevotella copri(P-enterotype).Significant less Bacteroides uniformis was found in the BB-enterotype patient group,while significant more butyrate-producing bacteria(e.g.Eubacterium rectale and Faecalibacterium prausnitzii)were found in the P-enterotype patient group,following koumiss administration.After stopping koumiss consumption,the relative abundance of some biomarker taxa returned to the original level,suggesting that the gut microbiota modulatory effect was not permanent and that continuous koumiss administration was required to maintain the therapeutic effect.In conclusion,koumiss consumption could alleviate the symptoms of CAG patients.Our results may help understand the mechanism of koumiss in alleviating CAG disease symptoms,facilitating the development of such products with desired therapeutic functions.展开更多
Portunus trituberculatus is an ide al model for elucidating crustacean genetic networks.Here we combined single molecule real-time(SMRT)sequencing and Illumina RNA-seq to characterize the coding genes,non-coding RNAs ...Portunus trituberculatus is an ide al model for elucidating crustacean genetic networks.Here we combined single molecule real-time(SMRT)sequencing and Illumina RNA-seq to characterize the coding genes,non-coding RNAs and pseudogenes and further to improve the genome annotation information of P.tritub erculatus.In this study,we assembled 9694 non-redundancy full-length transcripts,and 658737307-bp repetitive sequences were identified in the P.trituberculatus full-length transcriptome.We also predicted the P.tritub erculatus genome structure based on full-length transcripts,including 18602 genes,28686 non-coding RNAs,1407 pseudogenes,740 motif,and 26434 domain.Meanwhile,14460,10211,5412,7314,and 14448 genes had significant matches with sequences in the NR,KOG,GO,KEGG,and TrEMBL database,respectively.Overall,our work firstly provided the long-read transcriptome and we believed that these data are very necessary to improve the annotation information of P.trituberculatus genome structure,and useful information for the future studies on evolution and physiological regulation of P.trituberculatus.展开更多
Conventional filling therapy fails to fundamentally reduce oral cariogenic bacteria.Thus,oral microbiota follow-up intervention after filling would be necessary for improving dental caries prognosis.We recruited 9 car...Conventional filling therapy fails to fundamentally reduce oral cariogenic bacteria.Thus,oral microbiota follow-up intervention after filling would be necessary for improving dental caries prognosis.We recruited 9 caries-free individuals,and 89 dental caries subjects(5 dropouts).Eighty-nine patients were randomized into three groups:caries(n=8;no treatment),control(n=40;filling),and postbiotics(n=41;filling and 14-day Probio-Eco®intervention).Salivary samples were collected at 0 day(after filling)and 14 days.Our results showed that the diversity of dental caries oral microbiota was significantly increased compared with healthy subjects,and filling could restore a healthier oral microbiota partially and temporarily.Thepostbiotics intervention keeps a low alpha-diversity.Co-occurrence network analysis showed that a more stable oral microbiota structure after postbiotics intervention.Taxonomic and functional annotation of the microbiota revealed that postbiotics co-treatment significantly:increased the relative abundance of Pseudomonas and P.reactans,decreased the relative abundance of Prevotella shahii,and enriched the energy metabolism-related pathways.BugBase-predicted phenotypes inferred to an oral microbiota with decreased potential pathogenic bacteria and increased oxidative stress-tolerant bacteria after postbiotics intervention.Collectively,it suggested that postbiotics co-treatment could be a promising strategy that restores the oral microecological balance for dental caries.展开更多
The gut microbiota of Mongolian hosts has distinctive characteristics due to their meat- and dairyoriented daily diets and unique genotype.The aim of the present study was to investigate the effect of switching from t...The gut microbiota of Mongolian hosts has distinctive characteristics due to their meat- and dairyoriented daily diets and unique genotype.The aim of the present study was to investigate the effect of switching from the typical high protein and fat Mongolian diets to carbohydrate-rich meals composed principally of wheat,rice and naked oats on the host gut microbiota within 3 weeks.Our study took the advantage of the long sequence reads produced by the Pac Bio single molecule real-time sequencing technology to enable the profiling of subjects' gut microbiota communities along the diet intervention to the species precision.We found that the bacterial richness and diversity decreased apparently along the diet intervention.During the diet intervention,the gut microbiota composition displayed no significant difference at phylum level(with major phyla of Firmicutes,Bacteroidetes,Tenericutes and Proteobacteria).The relative abundances of some genera such as Bacteroidetes,Faecalibacterium,Roseburia,Alistipes,Streptococcus,and Oscillospira were significantly altered after the diet switching started.Notably,significant changes were also observed in the proportions of the species Bacteroides dorei,Bacteroides fragilis,Bacteroides thetaiotaomicron,Ruminococcus albus,Ruminococcus faecis,Roseburia faecis and Eubacterium ventriosum.These results have demonstrated that diet and host gut microbiota is closely linked.展开更多
文摘Single molecular real-time(SMRT)sequencing,also called third-generation sequencing,is a novel sequencing technique capable of generating extremely long contiguous sequence reads.While conventional short-read sequencing cannot evaluate the linkage of nucleotide substitutions distant from one another,SMRT sequencing can directly demonstrate linkage of nucleotide changes over a span of more than 20 kbp,and thus can be applied to directly examine the haplotypes of viruses or bacteria whose genome structures are changing in real time.In addition,an error correction method(circular consensus sequencing)has been established and repeated sequencing of a single-molecule DNA template can result in extremely high accuracy.The advantages of long read sequencing enable accurate determination of the haplotypes of individual viral clones.SMRT sequencing has been applied in various studies of viral genomes including determination of the full-length contiguous genome sequence of hepatitis C virus(HCV),targeted deep sequencing of the HCV NS5A gene,and assessment of heterogeneity among viral populations.Recently,the emergence of multi-drug resistant HCV viruses has become a significant clinical issue and has been also demonstrated using SMRT sequencing.In this review,we introduce the novel third-generation PacBio RSII/Sequel systems,compare them with conventional next-generation sequencers,and summarize previous studies in which SMRT sequencing technology has been applied for HCV genome analysis.We also refer to another long-read sequencing platform,nanopore sequencing technology,and discuss the advantages,limitations and future perspectives in using these thirdgeneration sequencers for HCV genome analysis.
基金supported by the National Natural Science Foundation of China (Grant No. 81373922)Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (Grant No. CIFMS, 2016-I2M-3–016)
文摘Global concerns have been paid to the potential hazard of traditional herbal medicinal products(THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, realtime(SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan(JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing(CCS) reads belonging to the ITS2 and psb A-trn H regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.
基金Supported by the National Natural Science Foundation of China(Nos.41006082,31372517)
文摘Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. ~To better understand the role of the surface-associated lifestyle of C. marina and the phylogeny of the family Halomonadaceae, we sequenced the entire genome of C. marina JCM 21022 ~T using single molecule real-time sequencing technology(SMR^T) and performed comparative genomics and phylogenomics analyses. ~The circular chromosome was 4 176 300 bp with an average GC content of 62.44% and contained 3 611 predicted coding sequences, 72 t RNA genes, and 21 r RNA genes. ~The C. marina JCM 21022 ~T genome contained a set of crucial genes involved in surface colonization processes. ~The comparative genome analysis indicated the significant diff erences between C. marina JCM 21022 ~T and Cobetia amphilecti KMM 296(formerly named C. marina KMM 296) resulted from sequence insertions or deletions and chromosomal recombination. Despite these diff erences, pan and core genome analysis showed similar gene functions between the two strains. ~The phylogenomic study of the family Halomonadaceae is reported here for the first time. We found that the relationships were well resolved among every genera tested, including Chromohalobacter, Halomonas, Cobetia, Kushneria, Zymobacter, and Halotalea.
文摘Intestinal dysbiosis is closely related to a variety of medical conditions,especially gastrointestinal diseases.The present study aimed to investigate the effects of koumiss on chronic atrophic gastritis(CAG)in an outpatient clinical trial(n=10;all female subjects aged 41-55;body mass index ranging from 19.5 to 25.8).Each patient consumed three servings of koumiss per day(i.e.250 ml daily before each of 3 meals)for a 60-day period.The improvement of patients'symptoms was monitored by comparing the total scores of symptoms before and after the treatment.Meanwhile,the changes in the patients’fecal microbiota composition and specific blood parameters were determined.After the 60-day koumiss administration,significant symptom improvements were observed,as evidenced by the reduction of the total symptoms score,and changes in blood platelet and cholesterol levels.The changes in patients’fecal microbiota composition were found.The patients’fecal microbiota fell into two distinct enterotypes,Bacteroides dorei/Bacteroides uniformis(BB-enterotype)and Prevotella copri(P-enterotype).Significant less Bacteroides uniformis was found in the BB-enterotype patient group,while significant more butyrate-producing bacteria(e.g.Eubacterium rectale and Faecalibacterium prausnitzii)were found in the P-enterotype patient group,following koumiss administration.After stopping koumiss consumption,the relative abundance of some biomarker taxa returned to the original level,suggesting that the gut microbiota modulatory effect was not permanent and that continuous koumiss administration was required to maintain the therapeutic effect.In conclusion,koumiss consumption could alleviate the symptoms of CAG patients.Our results may help understand the mechanism of koumiss in alleviating CAG disease symptoms,facilitating the development of such products with desired therapeutic functions.
基金Supported by the National Key Research and Development Program of China(No.2017YFA0604904)the Zhejiang Provincial Natural Science Foundation of China(No.LR21D060003)。
文摘Portunus trituberculatus is an ide al model for elucidating crustacean genetic networks.Here we combined single molecule real-time(SMRT)sequencing and Illumina RNA-seq to characterize the coding genes,non-coding RNAs and pseudogenes and further to improve the genome annotation information of P.tritub erculatus.In this study,we assembled 9694 non-redundancy full-length transcripts,and 658737307-bp repetitive sequences were identified in the P.trituberculatus full-length transcriptome.We also predicted the P.tritub erculatus genome structure based on full-length transcripts,including 18602 genes,28686 non-coding RNAs,1407 pseudogenes,740 motif,and 26434 domain.Meanwhile,14460,10211,5412,7314,and 14448 genes had significant matches with sequences in the NR,KOG,GO,KEGG,and TrEMBL database,respectively.Overall,our work firstly provided the long-read transcriptome and we believed that these data are very necessary to improve the annotation information of P.trituberculatus genome structure,and useful information for the future studies on evolution and physiological regulation of P.trituberculatus.
基金approved by the Medical Ethics Committee of Affiliated Stomatological Hospital of Nanchang University(2019015).
文摘Conventional filling therapy fails to fundamentally reduce oral cariogenic bacteria.Thus,oral microbiota follow-up intervention after filling would be necessary for improving dental caries prognosis.We recruited 9 caries-free individuals,and 89 dental caries subjects(5 dropouts).Eighty-nine patients were randomized into three groups:caries(n=8;no treatment),control(n=40;filling),and postbiotics(n=41;filling and 14-day Probio-Eco®intervention).Salivary samples were collected at 0 day(after filling)and 14 days.Our results showed that the diversity of dental caries oral microbiota was significantly increased compared with healthy subjects,and filling could restore a healthier oral microbiota partially and temporarily.Thepostbiotics intervention keeps a low alpha-diversity.Co-occurrence network analysis showed that a more stable oral microbiota structure after postbiotics intervention.Taxonomic and functional annotation of the microbiota revealed that postbiotics co-treatment significantly:increased the relative abundance of Pseudomonas and P.reactans,decreased the relative abundance of Prevotella shahii,and enriched the energy metabolism-related pathways.BugBase-predicted phenotypes inferred to an oral microbiota with decreased potential pathogenic bacteria and increased oxidative stress-tolerant bacteria after postbiotics intervention.Collectively,it suggested that postbiotics co-treatment could be a promising strategy that restores the oral microecological balance for dental caries.
文摘The gut microbiota of Mongolian hosts has distinctive characteristics due to their meat- and dairyoriented daily diets and unique genotype.The aim of the present study was to investigate the effect of switching from the typical high protein and fat Mongolian diets to carbohydrate-rich meals composed principally of wheat,rice and naked oats on the host gut microbiota within 3 weeks.Our study took the advantage of the long sequence reads produced by the Pac Bio single molecule real-time sequencing technology to enable the profiling of subjects' gut microbiota communities along the diet intervention to the species precision.We found that the bacterial richness and diversity decreased apparently along the diet intervention.During the diet intervention,the gut microbiota composition displayed no significant difference at phylum level(with major phyla of Firmicutes,Bacteroidetes,Tenericutes and Proteobacteria).The relative abundances of some genera such as Bacteroidetes,Faecalibacterium,Roseburia,Alistipes,Streptococcus,and Oscillospira were significantly altered after the diet switching started.Notably,significant changes were also observed in the proportions of the species Bacteroides dorei,Bacteroides fragilis,Bacteroides thetaiotaomicron,Ruminococcus albus,Ruminococcus faecis,Roseburia faecis and Eubacterium ventriosum.These results have demonstrated that diet and host gut microbiota is closely linked.
