| Background and Objectives:The aim of this study is to explore the research effects of different reduction methods of host nucleic acid applied to the virome and metagenomics of plasma,and to develop a complete reduction strategy,thereby improving the sequencing efficiency of Metagenomics Next-generation Sequencing(mNGS),increasing the types and quantities of detectable pathogens,and providing theoretical basis for the development of plasma pathogen screening strategies.Methods:(1)Strategies for host nucleic acid reduction in the virome of plasma:Pseudorabies virus(PRV),Porcine parvovirus(PPV),and Encephalomyocarditis virus(EMCV),which did not exist in normal human plasma,were used as indicator viruses and quantitatively injected into the plasma of unpaid blood donors scrapped due to high alanine transaminase(ALT),which was used as a prefabricated plasma sample.Simultaneously,human mitochondrial DNA(mtDNA)was selected to reflect changes in human nucleic acid in prefabricated plasma samples.Then the prefabricated plasma samples were treated with three kinds of library construction(the procedures of DNA library construction,RNA library construction and combined library construction),five kinds of centrifugation conditions(4℃,100g,30min;4℃,5000g,10min;4℃,5000g,15min;4℃,4000g,45min;4℃,17000g,3min),two kinds of filters(0.22μm and 0.45μm),four kinds of enzymes(deoxyribonuclease Ⅰ,ribonuclease A from bovine pancreas,benzonase nuclease and lysozyme)and four concentrations of chloroform(1%,5%,10%and 20%),respectively.The effects of reduction of host nucleic acid were evaluated by the changes in Ct values of mtDNA and indicator virus groups,the proportion of human and microbial sequences,and species richness.Finally,various host nucleic acid reduction methods with relatively ideal effects were formed into host nucleic acid reduction strategies to treat the prefabricated plasma samples.Subsequently,nucleic acid extraction,quantification by quantitative Realtime PCR(qPCR),and library construction were performed to compare their effectiveness.(2)Strategies for host nucleic acid reduction in the metagenomics of plasma:Escherichia coli,Staphylococcus aureus,and Staphylococcus epidermidis were quantitatively injected into human plasma as an indicator bacterium,which was as an experimental sample,and mtDNA was also selected to indicate changes in human nucleic acids.The experimental samples were treated by method related to saponin,optimized method related to saponin,simple centrifugation,centrifugation and washing,and nuclease,respectively.The effects of reducing host nucleic acid were evaluated by the Ct values of mtDNA and indicator bacteria,the proportion of human and microbial sequences,and the changes in species richness.Finally,the optimized method related to saponin was selected as the reduction strategy of host nucleic acid in metagenomics,and six concentration gradients of experimental samples formed after continuous gradient dilution of 10 times by plasma were processed to test the performance of this strategy.Results:(1)Strategies for host nucleic acid reduction in the virome of plasma:the procedures of DNA library construction,RNA library construction and combined library construction were applicable to the composition and properties research of dsDNA virus,RNA virus group and overall virus group respectively.Centrifugation at 4℃,5000g,10min was suitable for increasing the overall abundance of virus,and centrifugation at 4℃,100g,30min was suitable for increasing the content of virus similar to PRV.0.45-μm filter,DNase I,Benzonase and low concentration chloroform effectively reduced the proportion of non-viral nucleic acid sequence in plasma to increase the virus abundance.Five kinds of reduction strategies for depleting host nucleic acid were established to treat the prefabricated plasma samples by centrifugation at 4℃,5000g,10min,filtration with 0.45μm filter,1%chloroform and two nucleases(DNase I and Benzonase).The five strategies established not only reduced the concentration and the proportion of human sequences,but also reduced the proportion and relative abundance of microorganisms and indicator viruses,increased the number of unknown sequences,and had similar microbial composition to the blank control.(2)Strategies for host nucleic acid reduction in the metagenomics of plasma:both methods related to saponin decreased the concentration and proportion of human nucleic acid,elevated the proportion of microbial sequences and the abundances of S.aureus and S.epidermidis,and decreased the abundance of E.coli.There were no statistical differences in the effects of simple centrifugation,or centrifugation and washing on either host or indicator bacteria.Combined treatment with two nucleases,DNase I and benzonase,reduced the proportion of human sequences and microbial sequences,and the concentration and relative abundance of S.aureus and S.epidermidis,but substantially increased the relative abundance of E.coli.The optimized method related to saponin showed good linearity between the concentrations of the three bacteria after treating 6 gradients and the concentrations before treatment,with correlation coefficients(R2)all above 0.99 and good repeatability;the proportion of human sequences in gradients 1 to 3 was reduced,the proportion of microbial sequences was increased,and the three bacterial species in three concentration gradients were relatively enriched.Conclusions:The effectiveness of different reduction methods of host nucleic acid applied to the study of the virome and the metagenomics in plasma is various,and continuous optimization and validation are needed.Although the five reduction strategies of host nucleic acid applied to the virome in this study can achieve the goal of reducing host nucleic acid,they also reduce the virus and need further optimization.The established optimized method related to saponin may be applied as an ideal strategy in research of the metagenomics. |
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