Study And Application Of The Diversity Of Flies Carrying Bacteria Based On The Second-Generation Sequencing Technology

ObjectiveFlies are an important medical vector organism that carries a large number of bacterial pathogens.They can directly or indirectly transmit diseases and have a close relationship with human health.Therefore,detection of flies carrying bacteria is of great significance in preventing the occurrence and prevalence of corresponding diseases.At present,the detection efficiency of flies carrying bacteria detection methods is relatively low,the species of bacteria that could be detected are very limited,and it is impossible to reflect the actual conditions of the samples carrying bacteria.The Second-Generation Sequencing Technology has a high sequencing throughput.It could obtain all the nucleic acid sequences from the sample by one sequencing,and providing a possibile complete detection method for the flies carrying bacteria.In this study,the Second-Generation Sequencing Technology was used to study the diversity of flies carrying bacteria,aiming to establish an efficient method for the detection of flies carrying bacteria.MethodsResearch content is divided into four parts.In the first part,on the basis of the shaking elution method,the method of separation and collection of flies carrying bacteria was optimized,and two conditions affecting the collection efficiency were explored,namely the number of elution times and the amount of eluent collected.Through a single-factor controlled variable method,with the collection efficiency of bacterial DNA as an indicator,combined with the cumbersome degree of actual operation,the optimal elution times and eluent collection amount were obtained,resulting in a simple and highly efficient separation methods of flies carrying bacteria.In the second part,six methods of bacterial DNA extraction have been compared,including: magnetic method,pyrolysis method,DNA extraction kit method(with lysozyme treatment or without lysozyme treatment),ultrasonic method and ultrasonic with pyrolysis method.Using ten kinds of equal mixture of bacteria as a sample,their bacterial DNA was extracted by six methods,and the V3-V4 region of the 16 S rRNA gene of the bacteria was specifically amplified,and the PCR products were sequenced for the Second-Generation.By comparing the extraction efficiency of the six methods,the analysis content includes: the quality of the extracted bacterial DNA,the quality of the PCR product,and the degree of reaction of the sequencing result to the real situation of the sample(including the OTU abundance and the distribution of the number of bacteria),obtain the optimal method for bacterial DNA extraction.In the third part,a comparative study was conducted on the sequencing results of different regions of the 16 S rRNA gene of bacteria.The Chrysomya megacephala was used as an experimental sample and has been collected bacteria in vivo and in vitro respectively.After extracting the bacterial DNA,the V1-V3,V3-V4 and V4-V5 region genes of the 16 S rRNA of the bacteria were amplified by PCR,and the PCR amplification conditions were investigated.The PCR products were sequenced for the Second-Generation,then the PCR product quality and sequencing results(including data volume,species abundance,and diversity)of the three sequencing regions were compared and analyzed to obtain the most suitable Hypervariable area for sequencing.By analyzing the sequencing results of the bacteria carried in vivo and in vitro,the difference in the bacteria carried in vivo and in vitro is known.In the fourth part,the mixed bacteria solution and flies carrying bacteria were used as experimental samples to carry out Second-Generation Sequencing and Cloning-Sequencing analysis of the PCR products.A comparison analysis of both sequencing costs and sequencing results(including data volume,reproducibility,and species diversity)was conducted to understand the differences between Second-Generation Sequencing Technology and cloning-Sequencing.ResultsThe first part: When eluted 4 times,91.06% of bacteria can be collected;when the collected amount of eluent is all collected,the most bacteria could be collected,and the operation is simple.The second part: In addition to the low purity of bacterial DNA extracted by thermal pyrolysis method,the quality of bacterial DNA in the other five methods was better.The total amount of target fragments in the PCR products of pyrolysis method and ultrasonic with pyrolysis method was less than 1.5 ?g,which could not meet the requirements for Second-Sequencing library,and the remaining four methods were all satisfactory.In the Second-Generation Sequencing results,the OTU abundance of the magnetic method and the DNA extraction kit method(with lysozyme treatment)was 9 and the relative number of strains was the most evenly distributed.The third part: When the annealing temperature was 55°C,50°C and 48°C,the electrophoretic bands of PCR product of 16 S rRNA V1-V3,V3-V4 and V4-V5 were best.The PCR product obtained from the three V regions can meet the requirements of the Second-Generation Sequencing library.The best quality of PCR products in V3-V4 region,followed by V1-V3 region,and the worst amplification quality in V4-V5 region.The reproducibility of the in vivo sample was better than that of the in vitro sample,and the similarity of the dominant bacteria in the in vivo sample was higher than that of the in vitro sample,but the OTU abundance of the in vitro sample was larger than that of the in vivo sample.The fourth part: The amount of data produced by a single run of Second-Generation Sequencing far exceeds the number of CloningSequencing and the average cost of a single sequence does not exceed 0.2% of the Cloning-Sequencing.When the same bacterial DNA sample was analyzed,the results of Second-Generation Sequencing were more reproducible than the Cloning-Sequencing,and the species abundance was also much higher than the Cloning-Sequencing method.Conclusion1.While using shaking elution method to collect flies carrying bacteria,the best elution frequency was 4 times,and the best eluent collection amount was all collected.The experimental method for the separation and collection of fly samples carrying bacteria was obtained.2.Bacterial DNA extraction methods suitable for Second-Generation Sequencing Technologies are magnetic method and DNA extraction kit method(with lysozyme treatment).3.The optimal annealing temperatures for PCR amplified conditions of bacterial 16 S rRNA V1-V3,V3-V4 and V4-V5 were 55°C,50°C and 48°C,respectively,and suitable PCR amplification conditions were obtained..4.When 16 S rRNA was used as a molecular marker to study the diversity of flies carrying bacteria,selecting different hypervariable regions for sequencing had a certain impact on the results.The most suitable molecular marker for the diversity of flies carrying bacteria is the 16 S rRNA V3-V4 region gene,which has good sequencing quality and rich species information.5.The diversity of flies carrying bacteria in vivo is more stable than in vitro and will not change rapidly due to transfer to new environments.6.In bacterial diversity studies,the Second-Generation Sequencing is more cost-effective than the Cloning-Sequencing.