Comparison of different hypervariable regions of 16S rRNA for taxonomic profiling of vaginal microbiota using next-generation sequencing

2020 ◽  
Author(s):  
Auttawit Sirichoat ◽  
Nipaporn Sankuntaw ◽  
Chulapan Engchanil ◽  
Pranom Buppasiri ◽  
Kiatichai Faksri ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
Vaginal Microbiota ◽  
Next Generation ◽  
Taxonomic Profiling ◽  
Hypervariable Regions ◽  
Generation Sequencing

O-130 The influence of vaginal microbiota on frozen blastocyst implantation after transfer: a prospective study through next-generation 16S rRNA sequencing

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
H Asakura ◽  
Y Nakahara ◽  
Y Nagai ◽  
Y Sakuraba
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
Prospective Study ◽  
Embryo Implantation ◽  
Vaginal Microbiota ◽  
Vaginal Fluid ◽  
Next Generation ◽  
The Difference ◽  
A Prospective Study ◽  
Generation Sequencing

Abstract Study question A prospective study to investigate the relationship between the composition of vaginal microbiota through next-generation sequencing and the efficacy of single frozen blastocyst transfer in the same cycle. Summary answer Dominant presence of lactobacillus and other lactate producing microbes in the upper vagina was highly correlated with implantation of transferred blastocyst in this pilot study. What is known already Next-generation sequencing of 16S rRNA detected microbes in the uterine cavity and recent studies indicated that dominant presence of Lactobacillus correlated highly with successful implantation of the transferred embryos. Aberrant vaginal microbiota has been known to cause poor obstetrical outcomes, however little is known for its effect on embryo implantation in assisted reproduction. Study design, size, duration A prospective study with 25 female subjects transferring a frozen blastocyst using autologous oocyte, over 14 months period in 2019-2020. Participants/materials, setting, methods 25 female patients without tubal and uterine pathology and no history of multiple miscarriages and implantation failures were recruited with consent at a private ART clinic. Transdermal estrogen was used to prepare endometrium. Upper vaginal fluid was obtained in follicular phase of the the same cycle and analyzed through next-generation sequencing, but the result was reported after pregnancy confirmation. Single frozen blastocyst transfer and standard luteal phase support were performed. Institutional IRB approved the protocol. Main results and the role of chance The mean age was 36.2 y.o.(range 29-41 y.o.), and 14 gestational sacs (implantation rate 56%), and 3 miscarriage (21.4%) were observed. Next-generation sequencing for 16S rRNA revealed average 69.2% presence of Lactobacillus (0-100%) and average 78.0% (0.2-100%) lactate producing microbes (LPM: Lactobacillus, Bifidobacterium, Streptococcus, and Enterococcus) in the vaginal fluid. Using 90% as cut-off, implantation rates were 11/15 (73.3%) and 3/10 (30%) with Lactobacillus dominant and non-dominant, 12/16 (75%) and 2/9 (22.2%) with LPM dominant and non-dominant microbiota, respectively. The difference in each group were not statistically significant. The relative risks for pregnancy were 2.63 (95%CI 1.03-6.67, P=0.04) for Lactobacillus and 3.11 (95%CI 1.24-7.79, P=0.02) for LPM. As for ROC analysis for embryo implantation and dominant microbes, AUC and associated criterion were 0.62 and 90.7% (sensitivity 78.6%, specificity 72.7%) for Lactobacillus, 0.69 and 96.6% (sensitivity 85.7%, specificity 72.7%) for LPM, respectively. The difference of AUC was not significant (P=0.24). Limitations, reasons for caution Despite prospective nature of the study, small sample size limited the analytical power of the study. Aneuploidy screening was not performed to remove confounding factor. Wider implications of the findings Our pilot study revealed possible relationship between vaginal microbiota and embryo implantation. Dominance of Lactobacillus or other lactate producing microbes may be advantageous for successful ART. Sampling vaginal fluid for microbe analysis is less invasive than endometrial sampling and can obtain more abundant RNA with possible higher accuracy of analysis. Trial registration number not applicable

scholarly journals The Microbiota Profile Analysis of Combined Periodontal-Endodontic Lesions Using 16S rRNA Next-Generation Sequencing

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Ping Sun ◽  
Zhiyong Guo ◽  
Daiping Guo ◽  
Jian Wang ◽  
Tingting Wu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
Profile Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Periodontal Pocket ◽  
Rrna Gene ◽  
Next Generation ◽  
Linear Discriminant ◽  
Hypervariable Regions ◽  
Generation Sequencing

Objective. The primary aim of this investigation was to analyze the microbiome in patients with combined periodontal-endodontic lesions. Method. Patients with loose and/or painful teeth referred for treatment from March 2020 to December 2020 in the First People’s Hospital of Jinzhong were recruited. Samples were collected from teeth diagnosed as chronic periodontics (PE), ulcerative pulpitis (PU), and retrograde pulpitis (RE). Genomic DNA was extracted. The quantitative polymerase chain reaction, targeting the 16S ribosomal RNA (rRNA), was adopted for the quantification of bacteria. Then, the V3-V4 hypervariable regions of the 16S rRNA gene were amplified and subjected to next-generation sequencing. The statistical analysis was performed by R software (V3.5.1). Results. A total of 57 qualified samples were collected from 48 patients and analyzed (7 PE, 21 PU, and 19 RE). By linear discriminant analysis effect size, Kingella and Barnesiella were significantly increased in the periodontal pocket of retrograde pulpitis (RE-PE), compared with PE. The relative abundance of Clostridiales Incertae Sedis XI, Fusobacteriaceae, Fusobacterium, Parvimonas, Micrococcaceae, and Rothia was significantly increased in the pulp of retrograde pulpitis (RE-PU) than PU and RE-PE. Prevotella, Leptotrichia, Porphyromonas, Streptococcus, and Fusobacterium are consistently at a high abundance, across PU, RE-PE, and RE-PU. Conclusion. The current study highlighted the evidence that a specific microbial community is associated with the occurrence of retrograde pulpitis. The microenvironment of the root canal and pulp chamber will select microbiota. This study offered insights into the pathogenesis of retrograde pulpitis.

scholarly journals 16S rRNA Next Generation Sequencing Analysis Shows Bacteria in Alzheimer’s Post-Mortem Brain

2017 ◽  
Vol 9 ◽  
Author(s):  
David C. Emery ◽  
Deborah K. Shoemark ◽  
Tom E. Batstone ◽  
Christy M. Waterfall ◽  
Jane A. Coghill ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
Sequencing Analysis ◽  
Post Mortem ◽  
Next Generation ◽  
Post Mortem Brain ◽  
Next Generation Sequencing Analysis ◽  
Generation Sequencing

scholarly journals Descemet Membrane Endothelial Keratoplasty: NGS Metagenomics-Optimized Tissue Preparation and Surgical Technique. Doctoral Thesis

2021 ◽  
Author(s):  
◽  
Davide Borroni ◽  
Keyword(s):  
Next Generation Sequencing ◽  
Turnaround Time ◽  
Storage Conditions ◽  
Tissue Preparation ◽  
Next Generation ◽  
Human Donor ◽  
Taxonomic Profiling ◽  
Storage Media ◽  
Hypothermic Storage ◽  
Generation Sequencing

Title: Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium. Aim: To detect the presence of microorganisms in the storage media of human donor corneas using next-generation sequencing method. Methods: Seven samples from organ culture (OC) group (Cornea Max, Eurobio, Les Ulis, France) with one control (sterile media without any cornea) and seven samples from hypothermic storage group (Cornea Cold, Eurobio) with one control were used for this study. The corneas were placed in the respective storage media for 14 days before collecting the samples. Storage media (2 mL) from each sample were collected in RNAase-free tubes and shipped for ribosomal RNA sequencing of 16S and 18S. Simultaneously, another 1 mL of media sample was used for conventional diagnostic method (CDM) using Bactec instruments. Results: In both, OC and hypothermic storage and control samples, the most abundant genera were Pseudomonas, Comamonas, Stenotrophomonas, Alcanivorax, Brevundimonas and Nitrobacter. Acidovorax, Acetobacter and Hydrogenophilus were detected mostly in the hypothermic storage group. The most abundant fungal pathogen detected belonged to the genus Malassezia, which was found in both the storage conditions. CDM was negative for microorganisms in all the samples. Conclusion: Metagenomics provides full taxonomic profiling of the detected genomic material of the organisms and thus has the potential to deliver a much wider microbiological diagnostic approach than CDM. The costs and turnaround time need to be reduced, and; the detection of viable organisms would help this technology to be introduced into routine clinical practice.

scholarly journals Bacterial abundance and community composition in green, brown and red water from intensive catfish (Clarias sp.) culture ponds in Yogyakarta, Indonesia

2021 ◽  
Vol 22 (9) ◽  
Author(s):  
Karunia Adetera Nungki Wijayanti ◽  
Indah Istiqomah ◽  
Murwantoko Murwantoko
Keyword(s):  
Next Generation Sequencing ◽  
Bacterial Community ◽  
16S Rrna ◽  
Community Composition ◽  
Water Samples ◽  
Bacterial Abundance ◽  
Rrna Gene ◽  
Next Generation ◽  
Aquaculture System ◽  
Generation Sequencing

Abstract. Wijayanti KAN, Istiqomah I, Murwantoko. 2021. Bacterial abundance and community composition in green, brown and red water from intensive Catfish (Clarias sp.) culture ponds in Yogyakarta, Indonesia. Biodiversitas 22: 3677-3684. Catfish (Clarias sp.) is an important aquaculture commodity in Indonesia and cultured in an intensive system. Microorganisms play an important role in maintaining water quality of aquaculture system. The objective of this study was to determine the bacterial abundance and community composition of green, brown and red water collected from intensive catfish culture ponds in Yogyakarta using next-generation sequencing method. The water samples were collected from intensive catfish culture ponds with different colors, namely green, brown and red ponds located in Yogyakarta. The DNA from water samples was extracted using DNA extraction kit and used as template for 16S rRNA amplification. The V3-V4 hypervariable regions of the 16S rRNA gene were amplified apply for next-generation sequencing technology. This study could explore effectively the bacterial community in water samples. The bacterial communities in this catfish culture water showed higher bacterial richness compared to the other aquaculture system. The diversity of the green, brown and red catfish culture water ponds was similar with the number OTUs of the green, brown and red water samples, which were 1269; 1387 and 1323 OTUs respectively. The 694 OTUs (34.42%) were common core microbiomes in all catfish culture ponds, the 212 OTUs (10.51%) are present on green and brown water ponds, the 182 OTUs (9.02%) were on green and red water ponds, and the 183 OTUs (9.07%) were present on green and brown water ponds. However, the composition of the bacterial community was different. The most dominant phylum in green and brown water ponds was Proteobacteria with relative abundance in green water and brown water 71.6% and 47.0% respectively, whereas, the most dominant phylum in red water was Firmicutes (29.5%). The dominance of Firmicutes phylum in red water ponds may be caused by application of probiotic bacteria, the high organic content, and low oxygen concentration.

scholarly journals Application of Next-Generation Sequencing Technology Based on Single Gene Locus in Species Identification of Mixed Meat Products

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Xinmei Liu ◽  
Zhiyang Liu ◽  
Yiyu Cheng ◽  
Haijing Wu ◽  
Wei Shen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
16S Rrna ◽  
Species Identification ◽  
Growth Hormone Receptor ◽  
Single Gene ◽  
Meat Products ◽  
Quantitative Detection ◽  
Next Generation ◽  
Next Generation Sequencing Technology ◽  
Generation Sequencing

Polymerase chain reaction (PCR) detection is a commonly used method for species identification of meat products. However, this method is not suitable for the analysis of meat products containing multiple mixtures. This study aimed to test whether next-generation sequencing (NGS) technology could be used as a method for the certification of mixed meat products. In this study, five kinds of common meat (pigs, cattle, sheep, chickens, and ducks) were mixed as samples with different proportions. The primers designed from mitochondrial 16S rRNA and nuclear genome gene (growth hormone receptor, GHR), respectively, were used to detect these meats. The sequencing results of NGS were analyzed using a self-designed bioinformatics program. The fragments with similar sequences were classified and compared with the database to determine their species. The results showed that all five kinds of meat components could be correctly identified using these two primers. The meat composition could be detected as low as 0.5% in the mixed samples using the NGS technology targeting GHR gene fragments, which was superior to those targeting mitochondrial 16S rRNA. However, the quantitative detection of species in the mixture was not likely to be quite accurate due to the amplification bias of PCR amplification. These results showed that the NGS technology could be applied to identify meat species in mixtures.

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