2-Phase anaerobic digestion（AD）, where the acidogenic phase was operated at 2 day hydraulic retention time（HRT） and the methanogenic phase at 10 days HRT, had been evaluated to determine if it could provide higher...2-Phase anaerobic digestion（AD）, where the acidogenic phase was operated at 2 day hydraulic retention time（HRT） and the methanogenic phase at 10 days HRT, had been evaluated to determine if it could provide higher organic reduction and methane production than the conventional single-stage AD（also operated at 12 days HRT）. 454 pyrosequencing was performed to determine and compare the microbial communities. The acidogenic reactor of the 2-phase system yielded a unique bacterial community of the lowest richness and diversity, while bacterial profiles of the methanogenic reactor closely followed the single-stage reactor. All reactors were predominated by hydrogenotrophic methanogens, mainly Methanolinea. Unusually, the acidogenic reactor contributed up to 24%of total methane production in the 2-phase system. This could be explained by the presence of Methanosarcina and Methanobrevibacter, and their activities could also help regulate reactor alkalinity during high loading conditions through carbon dioxide production. The enrichment of hydrolytic and acidogenic Porphyromonadaceae, Prevotellaceae, Ruminococcaceae and unclassified Bacteroidetes in the acidogenic reactor would have contributed to the improved sludge volatile solids degradation, and ultimately the overall 2-phase system＇s performance. Syntrophic acetogenic microorganisms were absent in the acidogenic reactor but present in the downstream methanogenic reactor, indicating the retention of various metabolic pathways also found in a single-stage system. The determination of key microorganisms further expands our understanding of the complex biological functions in AD process.展开更多
BACKGROUND Kirsten rat sarcoma viral oncogene homolog(KRAS),neuroblastoma RAS viral oncogene homolog(NRAS),and v-raf murine sarcoma viral oncogene homolog B1(BRAF)nucleotide variants may generate quantitatively or qua...BACKGROUND Kirsten rat sarcoma viral oncogene homolog(KRAS),neuroblastoma RAS viral oncogene homolog(NRAS),and v-raf murine sarcoma viral oncogene homolog B1(BRAF)nucleotide variants may generate quantitatively or qualitatively various protein activities,which may be reflected in their differential association with tumor characteristics.AIM To examine the association between these mutations and colorectal cancer(CRC)progression stages.METHODS A retrospective analysis was conducted on 799 patients with CRC,whose tumor samples were examined for mutations in the hot-spots of the KRAS,NRAS,and BRAF genes at the University of Texas Medical Branch,spanning from January 2016 to July 2023.Statistical analyses were performed to assess the association of spe-cific nucleotide changes with tumor,nodes,and metastasis stages.RESULTS KRAS mutations were found in 39.5%of cases,NRAS mutations in 4.4%,and BRAF mutations in 6.0%.The KRAS p.Gly12Val and p.Gly13Asp mutations were positively associated with pathological stage 4 tumors.Additionally,the KRAS p.Gly12Asp and p.Gly12Val mutations were linked to an increased risk of distant metastasis.Meanwhile,the BRAF Val600Glu mutation was associated with a higher likelihood of lymph node involvement.CONCLUSION Our findings support the potential prognostic utility of specific KRAS(p.Gly12Val,p.Gly12Asp,and p.Gly13Asp)and BRAF p.Val600Glu mutations in CRC.These results are preliminary and require validation through larger,multi-center studies before they can be considered reliable in clinical practice.展开更多
AIM: To compare the sequencing of PCR products, pyro- sequencing, and real-time PCR for detection of Tyrosine- methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B. METHODS: Mixtures of p...AIM: To compare the sequencing of PCR products, pyro- sequencing, and real-time PCR for detection of Tyrosine- methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B. METHODS: Mixtures of plasmids and serum samples from 69 chronic hepatitis B patients treated with lamivu- dine were tested for YMDD mutations by sequencing of PCR products, pyrosequencing, and real-time PCR, re- spectively. Time required and reagent costs of the three assays were evaluated. RESULTS: Real-time PCR detected 100%, 50%, 10%, 1% and 0.1% of YVDD plasmid in mixtures with 106 copies/mL of YMDD plasmid, whereas sequencing and pyrosequencing only detected 100% and 50% of YVDD plasmid in aliquots of the corresponding mixtures. Com- pletely concordant results were obtained from 60 (87%) out of the 69 clinical serum samples by the three assays. Mutants were detected by real-time PCR in less than 20% of the total virus population, but no mutant was de- tected by sequencing and pyrosequencing. In addition, real-time PCR required less time and was more cost-ef- fective than the other two assays. However, throughput of pyrosequencing was the highest. CONCLUSION: Among the three assays compared, real-time PCR is the most sensitive, cost-effective, and time saving for monitoring YMDD mutants in patients with chronic hepatitis B on lamivudine therapy.展开更多
AIM:To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients.METHODS:Formalin-f ixed, paraff in-embedded (FFPE) sa...AIM:To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients.METHODS:Formalin-f ixed, paraff in-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 Chinese CRC patients at Beijing Cancer Hospital. After the extraction of genome DNA from FFPE samples, fragments contained codons 12 and 13 of KRAS exon 2 were amplified by polymerase chain reaction and analyzed by dideoxy sequencing, the KRAS Strip Assay and pyrosequencing. In addition, the sensitivities of the 3 methods were compared on serial dilutions (contents of mutant DNA: 100%,50%,20%, 5%,10%, 5%,1%,0%) of A549 cell line DNA (carrying the codon 12 Gly>Ser mutation) into wild-type DNA (human normal intestinal mucosa). The results of dideoxy sequencing,the KRAS StripAssay and pyrosequencing were analyzed by Chromas Software, Collector forKRAS Strip Assay and the pyrosequencing PyroMarkTM Q24 system, respectively.RESULTS: Among 100 patients, KRAS mutations were identif ied in 34%, 37% and 37% of patients by dideoxy sequencing, the KRAS StripAssay and pyrosequencing, respectively. The sensitivity was highest with the KRAS Strip Assay (1%), followed by pyrosequencing (5%), and dideoxy sequencing was lowest (15%). Six different mutation types were found in this study with 3 main mutations Gly12 Asp (GGT>GAT), Gly12 Val (GGT>GTT) and Gly13 Asp (GGC>GAC). Thirty-three patients were identifi ed to have KRAS mutations by the 3 methods, and a total of 8 patients had conflicting results between 3 methods: 4 mutations not detected by dideoxy sequencing and the KRAS StripAssay were identified by pyrosequencing; 3 mutations not detected by dideoxy sequencing and pyrosequencing were identif ied by the KRAS StripAssay; and 1 mutation not detected by pyrosequencing was conf irmed by dideoxy sequencing and the KRAS StripAssay. Among these discordant results, the results identif ied by dideoxy sequencing were consistent either with the KRAS StripAssay or with pyrosequencing, which indicated that the accuracy of dideoxy sequencing was high. CONCLUSION: Taking a worldwide view of reports and our results,dideoxy sequencing remains the most popular method because of its low cost and high accuracy.展开更多
The objective of this study was to investigate pig fed by Bacillus coagulans-fermented distillers＇ dried grains with solubles （DDGS） on the faecal microbial composition and diversity using 454 pyrosequencing. Healt...The objective of this study was to investigate pig fed by Bacillus coagulans-fermented distillers＇ dried grains with solubles （DDGS） on the faecal microbial composition and diversity using 454 pyrosequencing. Healthy crossbred （Durocx Yorkshirex Landrace） growing and fattening pigs （n=48）, with an average initial body weight of 65 kg, were divided into two groups （24 replicates per group; four pens per group; six pigs per pen）, and given either DDGS feed as the control, or B. coagulans-fermented DDGS feed as the treatment. Faecal samples were collected on day 0, 7, 14, 21, and 28. DNA was extracted, and the V3-V6 region of the 16S rRNA gene was amplified. The fermented DDGS feed affected the relative abundance of bacteria populations at the phylum, genus, and species levels. At the genus level, the consumption of fermented DDGS feed led to higher relative abundances of faecal Prevotella, Lactobacillus, Clostridium, Bifidobacterium, Roseburia, and Bacillus, and lower relative abundances of faecal Escherichia, Ruminococcus, Dialister, unclassified Lachnospiraceae, unclassified Ruminococcaceae, and unclassified Enterobacteriaceae than in the control. At the species level, the consumption of fermented DDGS feed led to higher relative abundances of faecal Prevotella sp., Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus mucosae, Lactobacillus reuteri, Clostridium butyricum, Bifidobacterium sp., and Roseburia sp., and lower relative abundances of faecal Prevotella copri, Escherichia coil, Ruminococcus gnavus, Ruminococcus flavefaciens, and Dialister sp. than in the control. Principal coordinates analysis indicated a distinct separation in the faecal microbial communities of pigs that were fed the fermented and unfermented DDGS feed. Fermented DDGS feed significantly increased the average daily gain （ADG） of pigs, and significantly decreased the average daily feed intake （ADFI） of feed and feed/gain （F/G）. Thus, our results demonstrate a beneficial shift in the faecal microbiota of pigs consuming fermented DDGS feed, with potential applications in livestock production.展开更多
The present study sampled the intestinal content of healthy and unhealthy Atlantic salmon(Salmo salar L.),the ambient water of unhealthy fish,and the biofilter material in the recirculating aquaculture system(RAS)to u...The present study sampled the intestinal content of healthy and unhealthy Atlantic salmon(Salmo salar L.),the ambient water of unhealthy fish,and the biofilter material in the recirculating aquaculture system(RAS)to understand differences in the intestinal microbiota.The V4--V5 regions of the prokaryotic 16S rRNA genes in the samples were analyzed by MiSeq high-throughput sequencing.The fish were adults with no differences in body length or weight.Representative members of the intestinal microbiota were identified.The intestinal microbiota of the healthy fish included Proteobacteria(44.33%),Actinobacteria(17.89%),Baeteroidetes(15.25%),and Firmicutes(9.11%),among which the families Mierococcaceae and Oxalobacteraceae and genera Sphingomonas,Streptomyces,Pedobacter,Janthinobacterium,Burkholderia,and Balneimonas were most abundant.Proteobacteria(70.46%),Bacteroidetes(7.59%),and Firmicutes(7.55%)dominated the microbiota of unhealthy fish,and Chloroflexi(2.71%),and Aliivibrio and Vibrio as well as genera in the family Aeromonadaceae were most strongly represented.Overall,the intestinal hindgut microbiota differed between healthy and unhealthy fish.This study offers a useful tool for monitoring the health status of fish and for screening the utility of probiotics by studying the intestinal microbiota.展开更多
This study aimed to reveal how amoxicillin（AMX） affected the microbial community and the spread mechanism of antibiotic resistance genes（ARGs） in the AMX manufacture wastewater treatment system. For this purpose, ...This study aimed to reveal how amoxicillin（AMX） affected the microbial community and the spread mechanism of antibiotic resistance genes（ARGs） in the AMX manufacture wastewater treatment system. For this purpose, a 1.47 L expanded granular sludge bed（EGSB） reactor was designed and run for 241 days treating artificial AMX manufacture wastewater. 454 pyrosequencing was applied to analyze functional microorganisms in the system. The antibiotic genes OXA_（-1）, OXA_（-2）, OXA_（-10）, TEM_（-1）, CTX-M_（-1）, class I integrons（intI1） and 16 SrRNA genes were also examined in sludge samples. The results showed that the genera Ignavibacterium, Phocoenobacter,Spirochaeta, Aminobacterium and Cloacibacillus contributed to the degradation of different organic compounds（such as various sugars and amines）. And the relative quantification of eachβ-lactam resistance gene in the study was changed with the increasing of AMX concentration.Furthermore the vertical gene transfer was the main driver for the spread of ARGs rather than horizontal transfer pathways in the system.展开更多
To understand the impacts of different plumbing materials on long-term biofilm formation in water supply system, we analyzed microbial community compositions in the bulk water and biofilms on faucets with two differen...To understand the impacts of different plumbing materials on long-term biofilm formation in water supply system, we analyzed microbial community compositions in the bulk water and biofilms on faucets with two different materials-polyvinyl chloride （PVC） and cast iron, which have been frequently used for more thanlO years. Pyrosequencing was employed to describe both bacterial and eukaryotic microbial compositions. Bacterial communities in the bulk water and biofilm samples were significantly different from each other. Specific bacterial populations colonized on the surface of different materials. Hyphomicrobia and corrosion associated bacteria, such as Acidithiobacillus spp., Aquabacterium spp., Limnobacter thiooxidans, and Thiocapsa spp., were the most dominant bacteria identified in the PVC and cast iron biofilms, respectively, suggesting that bacterial colonization on the material surfaces was selective. Mycobacteria and Legionella spp. were common potential pathogenic bacteria occurred in the biofilm samples, but their abundance was different in the two biofilm bacterial communities. In contrast, the biofilm samples showed more similar eukaryotic communities than the bulk water. Notably, potential pathogenic fungi, i.e., Aspergillus spp. and Candida parapsilosis, occurred in similar abundance in both biofilms. These results indicated that microbial community, especially bacterial composition was remarkably affected by the different pipe materials （PVC and cast iron）.展开更多
This study was to investigate bacterial and archaeal community structure of pan-Arctic Ocean sediments by pyrosequencing. In total, investigation of three marine sediments revealed 15 002 bacterial and 4 362 archaeal ...This study was to investigate bacterial and archaeal community structure of pan-Arctic Ocean sediments by pyrosequencing. In total, investigation of three marine sediments revealed 15 002 bacterial and 4 362 archaeal operational taxonomic units （OTUs） at the 97% similarity level. Analysis of community structure indicated that these three samples had high bacterial and archaeal diversity. The most relatively abundant bacterial group in Samples CC 1 and R05 was Proteobacteria, while Firmicutes was dominant in Sample BL03. Thaumarchaeota was the most relatively abundant archaeal phylum in Samples CC1 and R05, and the relative abundance of Thaumarchaeota was almost as high as that of Euryarchaeota in Sample BL03. These two phyla accounted for nearly 100% of the archaeal OTUs. 6-Proteobacteria and y-Proteobacteria were the two most relatively abundant classes at Proteobacterial class level, and their relative abundance was more than 60% in Samples CC1 and R05. There were also differences in the top 10 relatively abundant bacterial and archaeal OTUs among the three samples at the 97% similarity, and only 12 core bacterial OTUs were detected. Overall, this study indicated that there were distinct microbial communities and many unique OTUs in these three samples.展开更多
Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was ca...Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios (50∶1, 100∶1) and concentrations (13.0 pmol/25μL and 0.14 pmol/25μL, 19.5 pmol/25μL and 0.21 pmol/25μL), and the product yield and quality were compared respectively. Results The forward to reverse primer ratio of 50∶1 provided better yield and concentration of 19.5 pmol/25μL and 0.21 pmol//25μL generated a clearer band. Conclusion A simple and feasible method to produce HBV ssDNA for pyrosequencing in batch is established.展开更多
This study aimed to determine the microbial community structure of seawater in（ICE-1） and out（FUBIAO） of the pack ice zone in the Arctic region.Approximate 10 L seawater was filtrated by 0.2 μm Whatman nuclepore ...This study aimed to determine the microbial community structure of seawater in（ICE-1） and out（FUBIAO） of the pack ice zone in the Arctic region.Approximate 10 L seawater was filtrated by 0.2 μm Whatman nuclepore filters and the environmental genomic DNA was extracted.We conducted a detailed census of microbial communities by pyrosequencing.Analysis of the microbial community structures indicated that these two samples had high bacterial,archaeal and eukaryotic diversity.Proteobacteria and Bacteroidetes were the two dominant members of the bacterioplankton community in both samples,and their relative abundance were 51.29% and 35.39%,72.95%and 23.21%,respectively.Euryarchaeota was the most abundant archaeal phylum,and the relative abundance was nearly up to 100% in FUBIAO and 60% in ICE-1.As for the eukaryotes,no_rank_Eukaryota,Arthropoda and no_rank_Metazoa were the most abundant groups in Sample FUBIAO,accounting for 85.29% of the total reads.The relative abundance of the most abundant phylum in Sample ICE-1,no_rank_Eukaryota and no_rank_Metazoa,was up to 90.69% of the total reads.Alphaproteobacteria,Flavobacteria and Gammaproteobacteria were the top three abundant classes in the two samples at the bacterial class level.There were also differences in the top ten abundant bacterial,archaeal and eukaryotic OTUs at the level of 97% similarity between the two samples.展开更多
Based on the 454 pyrosequencing approach, this research evaluated the influence of coal mining subsi- dence on soil bacterial diversity and community structure in Chinese mining area. In order to characterize the bact...Based on the 454 pyrosequencing approach, this research evaluated the influence of coal mining subsi- dence on soil bacterial diversity and community structure in Chinese mining area. In order to characterize the bacterial community comparatively, this study selected a field experiment site with coal-excavated subsidence soils and an adjacent site with non-disturbed agricultural soils, respectively. The dataset com- prises 24512 sequences that are affiliated to the 7 phylogenetic groups： proteobacteria, actinobacteria, bacteroidetes, gemmatimonadetes, chlorofiexi, nitrospirae and unclassified phylum. Proteobacteria is the largest bacterial phylum in all samples, with a marked shift of the proportions of alpha-, beta-, and gammaproteobacteria. The results show that undisturbed soils are relatively more diverse and rich than subsided soils, and differences in abundances of dominant taxonomic groups between the two soil groups are visible. Compared with the control, soil nutrient contents decline achieves significant level in subsided soils. Correlational analysis showed bacterial diversity indices have significantly positive corre- lation with soil organic matter, total N, total P, and available K. but in negative relation with soil salinity. Ground subsidence noticeably affects the diversity and composition of soil microbial community. Degen- eration of soil fertility and soil salinization inhibits the sole-carbon-source metabolic ability of microbial community, leading to the simplification of advantage species and uneven distribution of microbial spe- cies. This work demonstrates the great potential of pyrosequencing technique in revealing microbial diversity and presents background information of microbial communities of mine subsidence land.展开更多
[Objective] The paper was to establish pyrosequencing methods for detecting viral hemorrhagic septicemia virus （VHSV）. [ Method ] One pair of PCR primers and one pyrosequencing primer of VHSV were designed. The pyro...[Objective] The paper was to establish pyrosequencing methods for detecting viral hemorrhagic septicemia virus （VHSV）. [ Method ] One pair of PCR primers and one pyrosequencing primer of VHSV were designed. The pyrosequencing reaction system and conditions were optimized and the pyrosequencing method for detecting VHSV was established. [ Result] This method was only able to specifically detect the objective viruses in the eight fish viruses, and the method had the advantage of high sensitivity. The minimum detectable limit of nucleic acid was 82 copies/μL. The method was verified by detecting VHSV in 1 924 batches of samples collected from domestic and imported fishes. The detection results were consistent with that of traditional RT-PCR, and the specificity and sensitivity of the method could meet the detection requirement for aquatic animal diseases. [ Conclusion] The study provides a new detection method for monitoring and prevention and control of aquatic animal virus diseases.展开更多
Sebastiscus marmoratus is an important sedentary ovoviparous fish distributed in near-shore coastal waters from the coast of China to Japan. Candidate S. marmoratus microsatellite markers were developed in the present...Sebastiscus marmoratus is an important sedentary ovoviparous fish distributed in near-shore coastal waters from the coast of China to Japan. Candidate S. marmoratus microsatellite markers were developed in the present study using 454 pyrosequencing, and the marker profile was analyzed. A total of 2 000 000 raw sequence reads were assembled to reduce redundancy. Among them, 1 043 dinucleotide, 925 trinucleotide, 692 tetranucleotide, and 315 pentanucleotide repeats were detected. AC repeats were the most frequent motifs among the dinucleotide repeats, and AAT was the most abundant among the trinucleotide repeats. AAAT, ATAG, and ATCC were the three most common tetranucleotide motifs, and AAGAT and AATAT were the most dominant pentanucleotide motifs. The greatest numbers of loci and potentially amplifiable loci were found in dinucleotide repeats, whereas trinucleotide repeats had the fewest. In summary, a wide range of candidate microsatellite markers were identified in the present study using a rapid and efficient 454 pyrosequencing approach.展开更多
Drinking water quality deteriorates from treatment plant to customer taps, especially in the plumbing system. There is no direct evidence about what the differences are contributed by plumbing system. This study compa...Drinking water quality deteriorates from treatment plant to customer taps, especially in the plumbing system. There is no direct evidence about what the differences are contributed by plumbing system. This study compared the water quality in the water main and at customer tap by preparing a sampling tap on the water main. The biomass was quantified by adenosine triphosphate(ATP) and the microbial community was profiled by 454 pyrosequencing.The results showed that in distribution pipes, biofilm contributed >94% of the total biomass,while loose deposits showed little contribution(< 2%) because of the low amount of loose deposits. The distribution of biological stable water had minor effects on the microbiocidal water quality regarding both quantity(ATP 1 ng/L vs. 1.7 ng/L) and community of the bacteria. Whereas the plumbing system has significant contribution to the increase of active biomass(1.7 ng/L vs. 2.9 ng/L) and the changes of bacterial community. The relative abundance of Sphingomonas spp. at tap(22%) was higher than that at water main(2%), while the relative abundance of Pseudomonas spp. in tap water(15%) was lower than that in the water from street water main(29%). Though only one location was prepared and studied, the present study showed that the protocol of making sampling tap on water main offered directly evidences about the impacts of plumbing system on tap water quality, which makes it possible to distinguish and study the processes in distribution system and plumbing system separately.展开更多
基金supported with funding from the National Research Foundation(NRF-CRP5-2009-02),Singapore for the project"Wastewater Treatment Plants as Urban Eco Power Stations"
文摘2-Phase anaerobic digestion（AD）, where the acidogenic phase was operated at 2 day hydraulic retention time（HRT） and the methanogenic phase at 10 days HRT, had been evaluated to determine if it could provide higher organic reduction and methane production than the conventional single-stage AD（also operated at 12 days HRT）. 454 pyrosequencing was performed to determine and compare the microbial communities. The acidogenic reactor of the 2-phase system yielded a unique bacterial community of the lowest richness and diversity, while bacterial profiles of the methanogenic reactor closely followed the single-stage reactor. All reactors were predominated by hydrogenotrophic methanogens, mainly Methanolinea. Unusually, the acidogenic reactor contributed up to 24%of total methane production in the 2-phase system. This could be explained by the presence of Methanosarcina and Methanobrevibacter, and their activities could also help regulate reactor alkalinity during high loading conditions through carbon dioxide production. The enrichment of hydrolytic and acidogenic Porphyromonadaceae, Prevotellaceae, Ruminococcaceae and unclassified Bacteroidetes in the acidogenic reactor would have contributed to the improved sludge volatile solids degradation, and ultimately the overall 2-phase system＇s performance. Syntrophic acetogenic microorganisms were absent in the acidogenic reactor but present in the downstream methanogenic reactor, indicating the retention of various metabolic pathways also found in a single-stage system. The determination of key microorganisms further expands our understanding of the complex biological functions in AD process.
文摘BACKGROUND Kirsten rat sarcoma viral oncogene homolog(KRAS),neuroblastoma RAS viral oncogene homolog(NRAS),and v-raf murine sarcoma viral oncogene homolog B1(BRAF)nucleotide variants may generate quantitatively or qualitatively various protein activities,which may be reflected in their differential association with tumor characteristics.AIM To examine the association between these mutations and colorectal cancer(CRC)progression stages.METHODS A retrospective analysis was conducted on 799 patients with CRC,whose tumor samples were examined for mutations in the hot-spots of the KRAS,NRAS,and BRAF genes at the University of Texas Medical Branch,spanning from January 2016 to July 2023.Statistical analyses were performed to assess the association of spe-cific nucleotide changes with tumor,nodes,and metastasis stages.RESULTS KRAS mutations were found in 39.5%of cases,NRAS mutations in 4.4%,and BRAF mutations in 6.0%.The KRAS p.Gly12Val and p.Gly13Asp mutations were positively associated with pathological stage 4 tumors.Additionally,the KRAS p.Gly12Asp and p.Gly12Val mutations were linked to an increased risk of distant metastasis.Meanwhile,the BRAF Val600Glu mutation was associated with a higher likelihood of lymph node involvement.CONCLUSION Our findings support the potential prognostic utility of specific KRAS(p.Gly12Val,p.Gly12Asp,and p.Gly13Asp)and BRAF p.Val600Glu mutations in CRC.These results are preliminary and require validation through larger,multi-center studies before they can be considered reliable in clinical practice.
文摘AIM: To compare the sequencing of PCR products, pyro- sequencing, and real-time PCR for detection of Tyrosine- methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B. METHODS: Mixtures of plasmids and serum samples from 69 chronic hepatitis B patients treated with lamivu- dine were tested for YMDD mutations by sequencing of PCR products, pyrosequencing, and real-time PCR, re- spectively. Time required and reagent costs of the three assays were evaluated. RESULTS: Real-time PCR detected 100%, 50%, 10%, 1% and 0.1% of YVDD plasmid in mixtures with 106 copies/mL of YMDD plasmid, whereas sequencing and pyrosequencing only detected 100% and 50% of YVDD plasmid in aliquots of the corresponding mixtures. Com- pletely concordant results were obtained from 60 (87%) out of the 69 clinical serum samples by the three assays. Mutants were detected by real-time PCR in less than 20% of the total virus population, but no mutant was de- tected by sequencing and pyrosequencing. In addition, real-time PCR required less time and was more cost-ef- fective than the other two assays. However, throughput of pyrosequencing was the highest. CONCLUSION: Among the three assays compared, real-time PCR is the most sensitive, cost-effective, and time saving for monitoring YMDD mutants in patients with chronic hepatitis B on lamivudine therapy.
文摘AIM:To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients.METHODS:Formalin-f ixed, paraff in-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 Chinese CRC patients at Beijing Cancer Hospital. After the extraction of genome DNA from FFPE samples, fragments contained codons 12 and 13 of KRAS exon 2 were amplified by polymerase chain reaction and analyzed by dideoxy sequencing, the KRAS Strip Assay and pyrosequencing. In addition, the sensitivities of the 3 methods were compared on serial dilutions (contents of mutant DNA: 100%,50%,20%, 5%,10%, 5%,1%,0%) of A549 cell line DNA (carrying the codon 12 Gly>Ser mutation) into wild-type DNA (human normal intestinal mucosa). The results of dideoxy sequencing,the KRAS StripAssay and pyrosequencing were analyzed by Chromas Software, Collector forKRAS Strip Assay and the pyrosequencing PyroMarkTM Q24 system, respectively.RESULTS: Among 100 patients, KRAS mutations were identif ied in 34%, 37% and 37% of patients by dideoxy sequencing, the KRAS StripAssay and pyrosequencing, respectively. The sensitivity was highest with the KRAS Strip Assay (1%), followed by pyrosequencing (5%), and dideoxy sequencing was lowest (15%). Six different mutation types were found in this study with 3 main mutations Gly12 Asp (GGT>GAT), Gly12 Val (GGT>GTT) and Gly13 Asp (GGC>GAC). Thirty-three patients were identifi ed to have KRAS mutations by the 3 methods, and a total of 8 patients had conflicting results between 3 methods: 4 mutations not detected by dideoxy sequencing and the KRAS StripAssay were identified by pyrosequencing; 3 mutations not detected by dideoxy sequencing and pyrosequencing were identif ied by the KRAS StripAssay; and 1 mutation not detected by pyrosequencing was conf irmed by dideoxy sequencing and the KRAS StripAssay. Among these discordant results, the results identif ied by dideoxy sequencing were consistent either with the KRAS StripAssay or with pyrosequencing, which indicated that the accuracy of dideoxy sequencing was high. CONCLUSION: Taking a worldwide view of reports and our results,dideoxy sequencing remains the most popular method because of its low cost and high accuracy.
基金the Open Funding Project of the Key Laboratory of Systems Bioengineering,Ministry of Education of China,Tianjin,China(20160315)
文摘The objective of this study was to investigate pig fed by Bacillus coagulans-fermented distillers＇ dried grains with solubles （DDGS） on the faecal microbial composition and diversity using 454 pyrosequencing. Healthy crossbred （Durocx Yorkshirex Landrace） growing and fattening pigs （n=48）, with an average initial body weight of 65 kg, were divided into two groups （24 replicates per group; four pens per group; six pigs per pen）, and given either DDGS feed as the control, or B. coagulans-fermented DDGS feed as the treatment. Faecal samples were collected on day 0, 7, 14, 21, and 28. DNA was extracted, and the V3-V6 region of the 16S rRNA gene was amplified. The fermented DDGS feed affected the relative abundance of bacteria populations at the phylum, genus, and species levels. At the genus level, the consumption of fermented DDGS feed led to higher relative abundances of faecal Prevotella, Lactobacillus, Clostridium, Bifidobacterium, Roseburia, and Bacillus, and lower relative abundances of faecal Escherichia, Ruminococcus, Dialister, unclassified Lachnospiraceae, unclassified Ruminococcaceae, and unclassified Enterobacteriaceae than in the control. At the species level, the consumption of fermented DDGS feed led to higher relative abundances of faecal Prevotella sp., Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus mucosae, Lactobacillus reuteri, Clostridium butyricum, Bifidobacterium sp., and Roseburia sp., and lower relative abundances of faecal Prevotella copri, Escherichia coil, Ruminococcus gnavus, Ruminococcus flavefaciens, and Dialister sp. than in the control. Principal coordinates analysis indicated a distinct separation in the faecal microbial communities of pigs that were fed the fermented and unfermented DDGS feed. Fermented DDGS feed significantly increased the average daily gain （ADG） of pigs, and significantly decreased the average daily feed intake （ADFI） of feed and feed/gain （F/G）. Thus, our results demonstrate a beneficial shift in the faecal microbiota of pigs consuming fermented DDGS feed, with potential applications in livestock production.
基金Supported by the National Natural Science Foundation of China(Nos.31472312,41306152,31402283)the Qingdao Innovation Talents Program(No.13-CX-16)+2 种基金the National Key Technology Research and Development Program of China(No.2014BAD08B09)the Earmarked Fund for Modern Agroindustry Technology Research System(No.CARS-48)the Project for International S&T Cooperation Program of China(No.2014DFA31030)
文摘The present study sampled the intestinal content of healthy and unhealthy Atlantic salmon(Salmo salar L.),the ambient water of unhealthy fish,and the biofilter material in the recirculating aquaculture system(RAS)to understand differences in the intestinal microbiota.The V4--V5 regions of the prokaryotic 16S rRNA genes in the samples were analyzed by MiSeq high-throughput sequencing.The fish were adults with no differences in body length or weight.Representative members of the intestinal microbiota were identified.The intestinal microbiota of the healthy fish included Proteobacteria(44.33%),Actinobacteria(17.89%),Baeteroidetes(15.25%),and Firmicutes(9.11%),among which the families Mierococcaceae and Oxalobacteraceae and genera Sphingomonas,Streptomyces,Pedobacter,Janthinobacterium,Burkholderia,and Balneimonas were most abundant.Proteobacteria(70.46%),Bacteroidetes(7.59%),and Firmicutes(7.55%)dominated the microbiota of unhealthy fish,and Chloroflexi(2.71%),and Aliivibrio and Vibrio as well as genera in the family Aeromonadaceae were most strongly represented.Overall,the intestinal hindgut microbiota differed between healthy and unhealthy fish.This study offers a useful tool for monitoring the health status of fish and for screening the utility of probiotics by studying the intestinal microbiota.
基金supported by the National Natural Science Foundation of China (No.51478138)the State Key Lab of Urban Water Resource and Environment (No.HIT ES200902)
文摘This study aimed to reveal how amoxicillin（AMX） affected the microbial community and the spread mechanism of antibiotic resistance genes（ARGs） in the AMX manufacture wastewater treatment system. For this purpose, a 1.47 L expanded granular sludge bed（EGSB） reactor was designed and run for 241 days treating artificial AMX manufacture wastewater. 454 pyrosequencing was applied to analyze functional microorganisms in the system. The antibiotic genes OXA_（-1）, OXA_（-2）, OXA_（-10）, TEM_（-1）, CTX-M_（-1）, class I integrons（intI1） and 16 SrRNA genes were also examined in sludge samples. The results showed that the genera Ignavibacterium, Phocoenobacter,Spirochaeta, Aminobacterium and Cloacibacillus contributed to the degradation of different organic compounds（such as various sugars and amines）. And the relative quantification of eachβ-lactam resistance gene in the study was changed with the increasing of AMX concentration.Furthermore the vertical gene transfer was the main driver for the spread of ARGs rather than horizontal transfer pathways in the system.
基金supported by the National Natural Science Foundation of China(No.51208501)the Key Research Program of the Chinese Academy of Sciences(No.KZZD-EW-09-1)the Program of the Chinese Academy of Sciences(No.Y025014EA2)
文摘To understand the impacts of different plumbing materials on long-term biofilm formation in water supply system, we analyzed microbial community compositions in the bulk water and biofilms on faucets with two different materials-polyvinyl chloride （PVC） and cast iron, which have been frequently used for more thanlO years. Pyrosequencing was employed to describe both bacterial and eukaryotic microbial compositions. Bacterial communities in the bulk water and biofilm samples were significantly different from each other. Specific bacterial populations colonized on the surface of different materials. Hyphomicrobia and corrosion associated bacteria, such as Acidithiobacillus spp., Aquabacterium spp., Limnobacter thiooxidans, and Thiocapsa spp., were the most dominant bacteria identified in the PVC and cast iron biofilms, respectively, suggesting that bacterial colonization on the material surfaces was selective. Mycobacteria and Legionella spp. were common potential pathogenic bacteria occurred in the biofilm samples, but their abundance was different in the two biofilm bacterial communities. In contrast, the biofilm samples showed more similar eukaryotic communities than the bulk water. Notably, potential pathogenic fungi, i.e., Aspergillus spp. and Candida parapsilosis, occurred in similar abundance in both biofilms. These results indicated that microbial community, especially bacterial composition was remarkably affected by the different pipe materials （PVC and cast iron）.
基金The Chinese Polar Environment Comprehensive Investigation and Assessment Program under contract No.CHINARE2014-03-05the National Natural Science Foundation of China under contract No.41176174the Innovation and Development Regional Demonstration Program of Marine Economy under contract No.12PYY001SF08-HYYS-1
文摘This study was to investigate bacterial and archaeal community structure of pan-Arctic Ocean sediments by pyrosequencing. In total, investigation of three marine sediments revealed 15 002 bacterial and 4 362 archaeal operational taxonomic units （OTUs） at the 97% similarity level. Analysis of community structure indicated that these three samples had high bacterial and archaeal diversity. The most relatively abundant bacterial group in Samples CC 1 and R05 was Proteobacteria, while Firmicutes was dominant in Sample BL03. Thaumarchaeota was the most relatively abundant archaeal phylum in Samples CC1 and R05, and the relative abundance of Thaumarchaeota was almost as high as that of Euryarchaeota in Sample BL03. These two phyla accounted for nearly 100% of the archaeal OTUs. 6-Proteobacteria and y-Proteobacteria were the two most relatively abundant classes at Proteobacterial class level, and their relative abundance was more than 60% in Samples CC1 and R05. There were also differences in the top 10 relatively abundant bacterial and archaeal OTUs among the three samples at the 97% similarity, and only 12 core bacterial OTUs were detected. Overall, this study indicated that there were distinct microbial communities and many unique OTUs in these three samples.
基金supported by the National Natural Science Foundation of China (No.60878056)the Doctoral Foundation of Xi’an Jiaotong University (DFXJTU2004-12)
文摘Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios (50∶1, 100∶1) and concentrations (13.0 pmol/25μL and 0.14 pmol/25μL, 19.5 pmol/25μL and 0.21 pmol/25μL), and the product yield and quality were compared respectively. Results The forward to reverse primer ratio of 50∶1 provided better yield and concentration of 19.5 pmol/25μL and 0.21 pmol//25μL generated a clearer band. Conclusion A simple and feasible method to produce HBV ssDNA for pyrosequencing in batch is established.
基金The National Natural Science Foundation of China under contract No.41176174the Chinese Polar Environment Comprehensive Investigation and Assessment Program under contract Nos CHINARE2013-03-05 and CHINARE 2014-03-05the Public Science and Technology Funds for Ocean Projects under contact No.201205020-5
文摘This study aimed to determine the microbial community structure of seawater in（ICE-1） and out（FUBIAO） of the pack ice zone in the Arctic region.Approximate 10 L seawater was filtrated by 0.2 μm Whatman nuclepore filters and the environmental genomic DNA was extracted.We conducted a detailed census of microbial communities by pyrosequencing.Analysis of the microbial community structures indicated that these two samples had high bacterial,archaeal and eukaryotic diversity.Proteobacteria and Bacteroidetes were the two dominant members of the bacterioplankton community in both samples,and their relative abundance were 51.29% and 35.39%,72.95%and 23.21%,respectively.Euryarchaeota was the most abundant archaeal phylum,and the relative abundance was nearly up to 100% in FUBIAO and 60% in ICE-1.As for the eukaryotes,no_rank_Eukaryota,Arthropoda and no_rank_Metazoa were the most abundant groups in Sample FUBIAO,accounting for 85.29% of the total reads.The relative abundance of the most abundant phylum in Sample ICE-1,no_rank_Eukaryota and no_rank_Metazoa,was up to 90.69% of the total reads.Alphaproteobacteria,Flavobacteria and Gammaproteobacteria were the top three abundant classes in the two samples at the bacterial class level.There were also differences in the top ten abundant bacterial,archaeal and eukaryotic OTUs at the level of 97% similarity between the two samples.
基金supported by the National Natural Science Foundation of China (No. 51174207)Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions of China (No. SZBF20116B35)
文摘Based on the 454 pyrosequencing approach, this research evaluated the influence of coal mining subsi- dence on soil bacterial diversity and community structure in Chinese mining area. In order to characterize the bacterial community comparatively, this study selected a field experiment site with coal-excavated subsidence soils and an adjacent site with non-disturbed agricultural soils, respectively. The dataset com- prises 24512 sequences that are affiliated to the 7 phylogenetic groups： proteobacteria, actinobacteria, bacteroidetes, gemmatimonadetes, chlorofiexi, nitrospirae and unclassified phylum. Proteobacteria is the largest bacterial phylum in all samples, with a marked shift of the proportions of alpha-, beta-, and gammaproteobacteria. The results show that undisturbed soils are relatively more diverse and rich than subsided soils, and differences in abundances of dominant taxonomic groups between the two soil groups are visible. Compared with the control, soil nutrient contents decline achieves significant level in subsided soils. Correlational analysis showed bacterial diversity indices have significantly positive corre- lation with soil organic matter, total N, total P, and available K. but in negative relation with soil salinity. Ground subsidence noticeably affects the diversity and composition of soil microbial community. Degen- eration of soil fertility and soil salinization inhibits the sole-carbon-source metabolic ability of microbial community, leading to the simplification of advantage species and uneven distribution of microbial spe- cies. This work demonstrates the great potential of pyrosequencing technique in revealing microbial diversity and presents background information of microbial communities of mine subsidence land.
基金Supported by the Twelfth Five-Year Support Project of the Ministry of Science and Technology(2013BAD12B02)Science and Technology Project of State General Administration of the People’s Republic of China for Quality Supervision and Inspection and Quarantine(2015IK195)
文摘[Objective] The paper was to establish pyrosequencing methods for detecting viral hemorrhagic septicemia virus （VHSV）. [ Method ] One pair of PCR primers and one pyrosequencing primer of VHSV were designed. The pyrosequencing reaction system and conditions were optimized and the pyrosequencing method for detecting VHSV was established. [ Result] This method was only able to specifically detect the objective viruses in the eight fish viruses, and the method had the advantage of high sensitivity. The minimum detectable limit of nucleic acid was 82 copies/μL. The method was verified by detecting VHSV in 1 924 batches of samples collected from domestic and imported fishes. The detection results were consistent with that of traditional RT-PCR, and the specificity and sensitivity of the method could meet the detection requirement for aquatic animal diseases. [ Conclusion] The study provides a new detection method for monitoring and prevention and control of aquatic animal virus diseases.
基金Supported by the Special Fund for Agro-scientific Research in the Public Interest(201303048)the National Natural Science Foundation of China(Nos.41176117,31172447)the National Infrastructure of Fishery Germplasm Resources
文摘Sebastiscus marmoratus is an important sedentary ovoviparous fish distributed in near-shore coastal waters from the coast of China to Japan. Candidate S. marmoratus microsatellite markers were developed in the present study using 454 pyrosequencing, and the marker profile was analyzed. A total of 2 000 000 raw sequence reads were assembled to reduce redundancy. Among them, 1 043 dinucleotide, 925 trinucleotide, 692 tetranucleotide, and 315 pentanucleotide repeats were detected. AC repeats were the most frequent motifs among the dinucleotide repeats, and AAT was the most abundant among the trinucleotide repeats. AAAT, ATAG, and ATCC were the three most common tetranucleotide motifs, and AAGAT and AATAT were the most dominant pentanucleotide motifs. The greatest numbers of loci and potentially amplifiable loci were found in dinucleotide repeats, whereas trinucleotide repeats had the fewest. In summary, a wide range of candidate microsatellite markers were identified in the present study using a rapid and efficient 454 pyrosequencing approach.
基金the support from the National Key R&D program of China(No.2018YFE0204100)the National Natural Science Foundation of China(No.52022103)the National Natural Science Foundation of China for International Cooperation and Exchange(No.51820105011)。
文摘Drinking water quality deteriorates from treatment plant to customer taps, especially in the plumbing system. There is no direct evidence about what the differences are contributed by plumbing system. This study compared the water quality in the water main and at customer tap by preparing a sampling tap on the water main. The biomass was quantified by adenosine triphosphate(ATP) and the microbial community was profiled by 454 pyrosequencing.The results showed that in distribution pipes, biofilm contributed >94% of the total biomass,while loose deposits showed little contribution(< 2%) because of the low amount of loose deposits. The distribution of biological stable water had minor effects on the microbiocidal water quality regarding both quantity(ATP 1 ng/L vs. 1.7 ng/L) and community of the bacteria. Whereas the plumbing system has significant contribution to the increase of active biomass(1.7 ng/L vs. 2.9 ng/L) and the changes of bacterial community. The relative abundance of Sphingomonas spp. at tap(22%) was higher than that at water main(2%), while the relative abundance of Pseudomonas spp. in tap water(15%) was lower than that in the water from street water main(29%). Though only one location was prepared and studied, the present study showed that the protocol of making sampling tap on water main offered directly evidences about the impacts of plumbing system on tap water quality, which makes it possible to distinguish and study the processes in distribution system and plumbing system separately.
