Studies On Phylogeny And Population Genetics Of Cryptosporidium Parvum ?d Subtype Family

Cryptosporidiosis has become one of the most important emerging zoonotic diseases in the world.It is considered to be the second leading cause of diarrhea and even death in children following rotavirus infection,and it is also the major cause of moderate-to-severe diarrhea in calves.Among the common species of Cryptosporidium that infect humans,C.parvum and C.hominis almost account for more than 90%of the causes of human cryptosporidiosis.The 60 kDa glycoprotein(GP60)gene is one of the most polymorphic gene loci in the Cryptosporidium genome,so it is usually used for the typing of C.parvum,C.hominis and other closely related species.In this study,literature retrieval was conducted on the two databases of PubMed and Web of Science through the keywords "Cryptosporidium","GP60" and "GP40/15",and sequence retrieval was conducted on GenBank,so as to download and sort out the relevant literature and sequences.Based on this,the contents related to the epidemiology of C.parvum were retrieved and the global epidemic situation was counted.And the global distribution of the ?a,?c and ?d subtype families of C.parvum and the distribution of each subtype of C.parvum in China were plotted by using geographic information system(GIS)software.The proportional similarity index(PSI)was then calculated to figure out the pattern of niche overlap(symbiosis)between countries.In this way,the global distribution of ?a,?c and ?d subtype family of C.parvum and the similarities and differences in terms of the population genetic structure of ?d subtype family in China and other countries were illustrated.This study combined single-cell whole genome amplification(WGA)technology with Oxford Nanopore long-read technology to explore a purification and WGA method suitable for a small number of Cryptosporidium samples.Firstly,1-2g of fresh stool samples were taken and degreased.After 1:1 sucrose solution gradient centrifugation and cesium chloride gradient centrifugation,the purified oocysts were selected by using a micromanipulator.Next,WGA products were then purified using AmPure XP magnetic beads after the oocysts were broken and single-cell WGA.Then the concentration and purity of the samples were tested and the qualified samples were constructed for database construction and the Oxford Nanopore long-read technology on the machine.Finally,the quality control and genome splicing were performed on the offline data.After the samples were purified and amplified,the DNA content reached ?g level from pg level after amplification,which ensured the purification quality and limited the loss within the tolerable range to meet the on-machine requirements of Nanopore sequencing technology(Nc/Qc?1.5,the total amount of sample? 5 ?g).After database construction and sequencing,the length of output data was mainly about 5 KB,reads N50 was between 6K and 7 K,and the GC content distribution of data was basically normal.The quality of sequenced reads was greater than that of Q7,and the sequencing quality was good.The size of the spliced genome was 9.08M,the N50 reached 1.11Mb,and the proportion of Complete BUSCOs reached 98.4%,indicating that the combination of this method was feasible.This study well solved the problems of the small size of some sample and the possiblely mixed infection in some samples,providing a new perspective for Cryptosporidium genome sequencing.A total of 53 positive samples of C.parvum ?d subtype family were collected from Henan and Xinjiang,including ?dA14G1(10),IIdA15Gl(11),IIdA19G1(22),and?dA20G1(10).After purification,the samples were first combined with the Next-generation Illumina sequencing technology by single cell isolation.Through qualitatively controlling the offline data and comparing with the reference genome for mutation detection and mutation annotation,and using genome-wide SNP data for population genetics research,the ML evolutionary tree was constructed and the principal component analysis and population structure analysis were also performed.It was found that the polymorphism of Cryptosporidium parvum was low in different regions of China,and genetic recombination occurred in some regions of the population.Finally,the Cryptosporidium-related database was constructed,including two modules of Genomics and GP60,and four tools of BLAST,Feature Search,JBrowse and FTP Download.This database system not only enriches Cryptosporidium-related data display methods,but also carries on the data retrieval,file management and the corresponding authorization management on this system.It can well meet the needs of research workers in the field of Cryptosporidium research in the process of data display,view and download.