Construction And Application Of Two Methods Of RNA Sequencing Libraries

?Objective?Disease prevention and control is the key to ensuring human and animal health.Viral infectious diseases have been produced for thousands of years,and harmed seriously survival and health of human and animal.Digging and identifying viruses and determining the relationship among viruses,diseases and hosts is a primary task in the prevention,diagnosis and treatment of viral infections.The next generation sequencing technology overcomes the limitations of traditional technology methods and satisfies the requirements of largescale sequencing tasks with high throughput,low cost and fast as the main features.It is the most efficient and accurate technologies for rapidly identifying and discovery unknown pathogens.Rapid detection of RNA viruses is critical for the diagnosis,treatment,control and prevention of human and animal infectious diseases caused by RNA viruses.In recent years,the development of next-generation sequencing(NGS)technologies has innovated methods for RNA virus detection.NGS can reveal a huge number of sequences of nucleic acids(DNA or RNA)within specimens through some random modes,and can detect various kinds of RNA viruses simultaneously thereby.This type of virome detection of RNA viruses has been widely applied to not only diagnosis of infectious diseases,but also identification of novel viruses and research of host-virus relationships.Because RNA has to be reverse transcribed before it can be sequenced using NGS technologies,and viral RNA in clinical specimens is usually limited in quantity and prone to degradation,efficient sequencing library preparation remains critical and challenging regarding virome detection of RNA viruses based on NGS technologies whose approach is still complex and needs to be optimized.In order to identify the RNA viruses better,we try to design a method to construct RNA virome sequencing librarythat is easy to use and has better sequencing results and costs less.The data and reference were provided for the construction of the next generation sequencing library of the RNA virome.?Methods?The first is to design the methods of next generation sequencing libraries prepared for the detection of viromes of RNA viruses.Method 1 was based on the commercial kit methodaccording to the manufacturer's instructions.Briefly,the extracted viral RNA was quantified,and then digested using RNase III.The fragmented RNA was ligated with random adaptors,and this was followed by reverse transcription.The c DNA was amplified through PCR,and the amplicons around 450 bp were collected.Method 2 was based on a procedure developed by ourselves.It began with random RT reaction,and specifically hydrolyzing RNA in the DNA-RNA heterozygous chain using RNase H after the first-strand c DNA synthesis.After the synthesis of the second-strand c DNA,library was amplified through PCR,and the amplicons around 450 bp were collected.Method 3 was modified from a procedure reported previously as sequence independent single primer amplification(SISPA).Briefly,the extracted viral RNA was reversely transcribed using the random primer SPN?8,and then the RT system was denaturated.The second-strand c DNA synthesis was modified using the enaturated RT system.This was followed with PCR using the primer SP(Table 1).The PCR amplicons were sheared and then ligated with sequencing adaptors.After that,the ligation products around 450 bp were collected and amplified.Method 3was modified from a procedure reported previously as sequence independent single primer amplification(SISPA).Briefly,the extracted viral RNA was reversely transcribed using the random primer SPN?8,and then the RT system was denaturated.The second-strand c DNA synthesis was modified using the denaturated RT system.This was followed with PCR using the primer SP.The PCR amplicons were sheared and then ligated with sequencing adaptors.After that,the ligation products around 450 bp were collected and amplified.After the successful design of the new methods,libraries were prepared for known samples using three methods respectively.All libraries were sequenced through the Ion Torrent PGM platformwith the same sequencing reagents.Each library was sequenced separately on an Ion 318? Chip.The advantages and disadvantages of the three methods were compared in terms of the number and type of reads and contigs sequenced,the depth of sequencing and the average coverage.Finally,the clinical samples were collected and sequenced by three methods to verify the characteristics of each method in the identification and characterization of the virus.?Results?The main contents and results of this thesis are as follows:(1)Construction of the next generation sequencing library of the RNA virome In the experiment,we used three methods to prepare the next generation sequencing library of RNA virome.In Method 1,the sequencing primers are introduced by random hybridization and linked to fragmented viral RNA using Ion Total RNA-Seq v2.Method 2 is designed by ourselves,whose principle is through random reverse transcription(RT)and polymerase chain reaction(PCR)to build the library.In addition,Method 3 uses a single primer to hybridize and ligation to a fragmented randomized RT-PCR amplification,which is improved by means of sequence independent single primer amplification(SISPA)method.In this study,we constructed sequencing libraries of the simulate sample containing specific AIV,NDV,and IBV which were detected by PCR on the collected clinical samples and sequenced at the same time to detecte the feasibility of Methods 2 and 3,and compared the three methods.In the experiment,it was found that the use of RNase III in method 1 makes it difficult to determine the time of RNA fragmentation and is difficult to control the quality of the library.The study found that all methods detected both AIV and NDV,whereas only Method 3 could detect IBV.Methods 2 and 3 were possibly superior to method 1 to be used for preparing the sequencing libraries for detection of viromes of RNA viruses.From the sequencing depth and the sequencing coverage analysis table,methods 2 and3 are superior to method 1 in terms of sequencing depth and sequencing coverage,and method 3 is superior to method 2 in sequencing coverage.(2)viral metagenomic analysis of apooled duck feces sample In this study,a pooled duck feces sample was collected from live poultry market,and libraries were prepared using three methods for sequencing,whose results were analyzed and compared.The study found that methods 2 and 3 were possibly superior to method 1 to be used for preparing the sequencing libraries for detection of viromes of RNA viruses.Method 1 has no advantage in detecting and identifying RNA viruses at low RNA concentration samples.Methods 2 and 3 can detect more viral families and viral genera,and method 2 is more advantageous.In addition,when analyzing the virus to ensure that most hits are reliable,we manually checkedall 843 viruses excluding phages and found that all virus hits had 30 exceptions for the virus hits.Through the analysis of the virus host,we found that there are some viruses in the living environment and diet of ducks,which can provide theoretical guidance for the prevention of potential diseases,food hygiene and disinfection of the environment and so on.(3)viral metagenomic analysis of apooled mink feces sample In this study,a pooled duck feces sample was collected from the mink farms,and libraries were prepared using three methods for sequencing,whose results were analyzed and compared.The study found that methods 2 and 3 could detect more viral families and viral genera,and that method 2 obtained the most viral contigs.Manually checked out all 3534 viruses excluding phages and found that all virus hits had 10 exceptions for the virus hits.Methods 2 and3 were superior to the method 1in the analysis of sequencing coverage of mink coronavirus strain WD1127 and WD1133,and method 2 had advantages over WD1127 strain,and methods had advantages over WD1133 strain.Throughanalysising virus hostand source,we found that some important mink virus data and multiple viruses may be reported firstly in this studyin mink,which provided a theoretical referencefor the detection and early warning of mink virus.?Conclusions? In summary,this study established reliable methods for the preparation of the next generation sequencing libraries of the RNA virome.We collected a mock sample containing three known viruses AIV,NDV and IBV,apooled duck feces sample and apooled mink feces sample,and constructed next generation sequencing library by three methods and sequenced whose results were analyzed and compared.Through the above study,we found that methods 2 and3 could btain more contigs mapped to viruses than method 1,and that more viral families and genera could be identified.Method 3 seems to be the best in sequencing depth and coverage analysis.Because both Method 1 and Method 3 require specific commercial kits for nucleic acid fragmentation and ligation of sequencing adapters,it is more expensive than method 2 that does not require any kits.From the ease of operation,methods 2 and 3 are simple and time-consuming to operate,and it is easy to control the quality of the sequencing libraries,in which the method 2 has moreadvantageous.This study is important for virus detection and produces important genomic data on ducks and minks(for example,many mink viruses are reported for the first time through this study).Because methods 1 and 3 require specific commercial kits for nucleic acid fragmentation and sequencing of joint connections,it is more expensive than the method without the need for any kit for the 2.From the degree of difficulty of operation,the method 2 and method of operation is simple and time-consuming,and easy to control the quality of the building,which is more advantage of the method of 2.In conclusion,this study is of significance for selecting proper methods to prepare sequencing libraries for viromes detection of RNA viruses.It also generated some important virome data regarding ducks and minks,and multiple viral genera were possibly first reported in minks through this study.