Pyrosequencing检测口腔与胃中的幽门螺杆菌16SrDNAV1区基因序列

目的通过Pyrosequencing检测口腔与胃幽门螺杆菌16SrDNAV1区序列基因片段分析,以进一步验证口—口传播的的假设。方法选择18例患有慢性中度牙周炎且胃镜活检Hp感染阳性患者及其中10例患者的家属。提取患者胃、牙菌斑和含漱液共74例样本中的DNA,PCR扩增阳性后pyrosequecing检测16SrDNAV1区序列基因片段。结果患者胃和口腔Hp及其家属口腔Hp的序列相比较,有0～1个碱基不同。结论口腔中的Hp与胃内Hp16SrDNAV1区基因型比较95.8%～100%的同源性,口腔可能为Hp的聚集地。口腔Hp可能与胃Hp感染的复发或再感染有关。

采用Pyrosequencing和Pooling技术对SNP位点多态性分析

目的建立基于pyrosequencing和Pooling技术进行SNP位点的法医学多态性分析技术。方法对50名无关个体样本建立一适合pyrosequencing检测的组池;采用PyroMark Assay Design 2.0软件进行SNP位点等位基因定量分析的引物设计;对组池样本PCR产物进行焦磷酸测序检测。结果检测的3个SNP位点多态性良好,其中位点rs220028与以往人群调查后频率数据无显著差异。结论采用pyrosequencing和Pooling技术对SNP位点进行多态性分析,适合于位点的初筛及大规模群体调查。该技术准确可靠,方便快捷。

Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B

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.

Comparative analysis of dideoxy sequencing,the KRAS StripAssay and pyrosequencing for detection of KRAS mutation

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.

Consuming fermented distillers＇ dried grains with solubles (DDGS) feed reveals a shift in the faecal microbiota of growing and fattening pigs using 454 pyrosequencing

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.

Bacterial and archaeal community structure of pan-Arctic Ocean sediments revealed by pyrosequencing

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.

Asymmetric PCR method in generation of HBV ssDNA for pyrosequencing

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.

Microbial community structure of Arctic seawater as revealed by pyrosequencing

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.

Pyrosequencing-based assessment of bacterial community structure in mine soils affected by mining subsidence

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.

Profile of candidate microsatellite markers in Sebastiscus marmoratus using 454 pyrosequencing

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.

Establishment of Pyrosequencing Technology for Detecting Viral Hemorrhagic Septicemia Virus (VHSV)

[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.

Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequencing now makes 16S rDNA assays more accessible to routine diagnostic laboratories, but this approach has had limited evaluation in general diagnostic practice. In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture. We found that use of the real-time 16S rDNA assay was clinically valuable in this specimen type because it enabled us to identify a small number of culture-negative infections. Although faster and less labour-intensive, we found that the utility of pyrosequencing for pathogen identification is still hampered by shorter read lengths compared to conventional (Sanger) sequencing. Combining results from both molecular and conventional culture methods, bacteria were only detected in 11.8% specimens in this study. However, the detection rate was increased to 18.6% if specimens were only included from patients with a documented clinical suspicion of infection. In conclusion, while pyrosequencing had significant advantages in speed and ease-of-use over conventional sequencing, multiple reactions will be required to deliver comparable species-level identification, thus negating many of the benefits of using the technique. We found that 16S rDNA PCR and sequencing should be rationally targeted on the basis of good clinical information in the routine diagnostic setting, and not used as a general screening test for the exclusion of bacterial infection in joint specimens.

Utilization of Pyrosequencing to Monitor the Microbiome Dynamics of Probiotic Treated Poultry (<i>Gallus gallus domesticus</i>) during Downstream Poultry Processing

Antibiotic growth promoters that have been historically employed to control pathogens and increase the rate of animal development for human consumption are currently banned in many countries. Probiotics have been proposed as an alternative to control pathogenic bacteria. Traditional culture methods typically used to monitor probiotic effects on pathogens possess significant limitations such as a lack in sensitivity to detect fastidious and non-culturable bacteria, and are both time consuming and costly. Here, we tested next generation pyrosequencing technology as a streamline and economical method to monitor the effects of a probiotic on microbial communities in juvenile poultry (Gallus gallus domesticus) after exposure to several microbiological challenges and litter conditions. Seven days and repeated again at 39 days following hatching, chicks were challenged with either Salmonella enterica serovar Enteritidis, Campylobacter jejuni, or no bacteria in the presence of, or without a probiotic (i.e., Bacillus subtilis) added to the feed. Three days following each of two challenges (i.e., days 10 and 42, respectively) the microbiome distributions of the poultry caecum were characterized based on 16S rDNA analysis. Generated PCR products were analyzed by automated identification of the samples after pooling, multiplexing and sequencing. A bioinformatics pipeline was then employed to identify microbial distributions at the phylum and genus level for the treatments. In conclusion, our results demonstrated that pyrosequencing technology is a rapid, efficient and cost-effective method to monitor the effects of probiotics on the microbiome of poultry propagated in an agricultural setting.

Analysis of PTEN Methylation Pattern by Pyrosequencing in Renal Cell Carcinoma

Objectives: We aimed to determine the frequency of PTEN (phosphatase with tensin homology deleted in chromosome 10) hypermethylation in renal cell carcinoma (RCC) and its impact on overall (OS) and disease-free survival (DFS). The association between PTEN hypermethylation and clinic and pathologic factors was also analyzed. Materials and Methods: The authors analyzed 137 patients who had undergone a radical or partial nephrectomy between 1997 and 2011. The methylation pattern was quantified individually at multiple CpG islands that are located on the exon 1 of PTEN gene by pyrosequencing. Results: Mean follow-up was 32.3 months. PTEN hypermethylation was detected with a low frequency (3.6%). There were no deaths or disease recurrence among patients with PTEN hypermethylation. Determining the association between PTEN hypermethylation and clinical and pathological factors was not possible due to the low frequency observed. Conclusion: The results show that the methylation of promoter CpG sites in PTEN is relatively infrequent in renal carcinogenesis and has no prognostic impact on survival.

Comparison of the Bacterial Microbiota in a Bale of Collected Cardboard Determined by 454 Pyrosequencing and Clone Library

Biofouling, the accumulation of microorganisms, is a major problem in paper mills processing paper and cardboard. This leads to the production of lower quality recycled products. Several studies have focused on the microbial content in the paper mill and the final products. Our aim was to determine the microbial biota in a bale of collected cardboard prior to entering the paper mill. Total genomic DNA was isolated and analyzed using two different methods for comparison purposes: 454 pyrosequencing and clone library. A total of 3268 V6-V8 454 pyrosequencing reads and 322 cloned V6-V8 16S rRNA nucleotide sequences were obtained. Both methods showed the presence of three major bacterial genera: Bacillus, Solibacillus and Paenibacillus, all members of the spore-forming phylum Firmicutes. Pyrosequencing, however, revealed a richer and more diverse bacterial community than clone library. It showed the presence of additional minor Firmicute genera and of a small number of Proteobacteria. The sorting at the recycling plant, the storing, and the processing at the paper mill, the end uses, will all contribute to the bacterial microbiota present in a bale of collected cardboard as revealed here.

TCERG1L hypermethylation is a risk factor of diabetic retinopathy in Chinese children with type 1 diabetes

●AIM:To identify the differential methylation sites(DMS)and their according genes associated with diabetic retinopathy(DR)development in type 1 diabetes(T1DM)children.●METHODS:This study consists of two surveys.A total of 40 T1DM children was included in the first survey.Because no participant has DR,retina thinning was used as a surrogate indicator for DR.The lowest 25%participants with the thinnest macular retinal thickness were included into the case group,and the others were controls.The DNA methylation status was assessed by the Illumina methylation 850K array BeadChip assay,and compared between the case and control groups.Four DMS with a potential role in diabetes were identified.The second survey included 27 T1DM children,among which four had DR.The methylation patterns of the four DMS identified by 850K were compared between participants with and without DR by pyrosequencing.●RESULTS:In the first survey,the 850K array revealed 751 sites significantly and differentially methylated in the case group comparing with the controls(|Δβ|>0.1 and Adj.P<0.05),and 328 of these were identified with a significance of Adj.P<0.01.Among these,319 CpG sites were hypermethylated and 432 were hypomethylated in the case group relative to the controls.Pyrosequencing revealed that the transcription elongation regulator 1 like(TCERG1L,cg07684215)gene was hypermethylated in the four T1DM children with DR(P=0.018),which was consistent with the result from the first survey.The methylation status of the other three DMS(cg26389052,cg25192647,and cg05413694)showed no difference(all P>0.05)between participants with and without DR.●CONCLUSION:The hypermethylation of the TCERG1L gene is a risk factor for DR development in Chinese children with T1DM.

Sanger和Pyrosequencing测序法分析健康成人口腔菌群

目的对比Sanger和Pyrosequencing测序法分析健康人口腔菌群组成。方法收集6例健康成人唾液、舌背、黏膜、龈上及龈下菌斑并构建16SrRNA基因文库,分别用Sanger和Pyrosequencing测序法分析。结果 Sanger测序所得已知的序列有5,794条(占6,535总序列数88.7%)、75个属,396个序列划分操作分类单元(operational taxonomic units,OTUs,占总OTUs的61.4%)。Pyrosequencing测序所得已知的序列有10,771条(占11,103总序列数97.0%)、66个属,322个OTUs(占总OTUs的68.0%)。Sanger和Pyrosequencing测序法所得口腔菌群在门、属的水平分布趋势基本一致,但在种的水平分布差异显著。Sanger和Pyrosequencing测序法构建的口腔菌群文库均匀度值分别为0.016和0.007,说明Pyrosequencing分析口腔菌群物种数量分布比Sanger测序方法的文库均匀性稍差,但优势种更显著。结论 Pyrosequencing测序时所构建基因文库能代表口腔菌群的多样性且经济、省时,可以应用于口腔细菌物种的分析。

Microbial community dynamics at high organic loading rates revealed by pyrosequencing during sugar refinery wastewater treatment in a UASB reactor

The performance and rnicrobial community structure in an upflow anaerobic sludge blanket reactor （UASB） treating sugar refinery wastewater were investigated. The chemical oxygen demand （COD） removal reached above 92.0% at organic loading rates （OLRs） of 12.0-54.0 kgCOD/（m^3· d）. The volatile lhtty acids （VFAs） in effluent were increased to 451.1 mg/L from 147.9 mg/L and the specific methane production rate improved by 1.2 2.2-1bid as the OLR increased. The evolution of microbial comnmnities in anaerobic sludge at three different OLRs was investigated using pyrosequencing. Operational taxonomic units （OTUs） at a 3% distance were 353,337 and 233 for OLRI2, OLR36 and OLR54, respectively. When the OLR was increased to 54.0 kgCOD/（m^3· d） from 12.0 kgCOD/ （m^3· d） by stepwise, the microbial community structure were changed significantly. Five genera （Bacteroides, Trichococcus, Cho,seobacterium, Longilinea and Aerococcus） were the dominant fermentative bacteria at the OLR 12-0 kgCOD/（m^3· d）. However, the sample of OLR36 was dominated by Lacmcoccus, Trichococcus, Anaer-arcus and Veillonella. At the last stage （OLR = 54.0 kgCOD/ （m^3· d）, the diversity and percentage of femlentative bacteria were markedly increased. Apart from fermentative bacteria, an obvious shift was observed in hydrogen-producing acetogens and non- acetotrophic methanogens as OLR increased. Svntrophohacter, Geobacter and Methanomethylovor- ans were the dominant hydrogen-producing acetogens and methylotrophic methanogens in the samples of OLRI2 and OLR36. When the OLR was increased to 54.0 kgCOD/（m^3· d）, the mare hydrogen-producing acetogens and hydrogenotrophic methanogens were substituted with Destd/bvi- brio and Methanospillum. However, the composition of acetotrophic methanogens （Methanosaeta） was relatively stable during the whole operation period of the UASB reactor.

Determination of the archaeal and bacterial communities in two-phase and single-stage anaerobic systems by 454 pyrosequencing

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.