The Preliminary Research Of Assembly Of Macaca Fascicularis MHC Genome With Fosmid Libraries

Cynomolgus macaques(Macaca fascicularis) are becoming a experimental animal for biomedical and biology research, because they are smaller and easier to manage, are more economical to maintain, and breed seasonally in captivity since the early 20 th century. The research of the cynomolgus macaque is of structural, functional, and evolutionary interest. As a vital factor in the determination of susceptibility and developing speed of disease, the structure and function of MHC has been the focus of exploration and research. And since MHC has an very high level of complexity with respect to sequence diversity and conservation and variability in the rates and types of gene duplications and indel activity, it is unusual difficult to reaserach. Our first study is to construct the genomic Fosmid libraries of Macaca fascicularis, and then screen the MHC contigs, assembly and Gene annotation of the MHC regions, and providing sequencing data of further gene function analysis. The main research results are as follows:(1)The genomic DNA used for constructing Fosmid library was extracted from peripheral venous blood of a male cynomolgus monkey with p CC2 FOS vector. And the Fosmid library consisting of 777,000 clones and saved in 312 poolings. The average insert size is calculated to be 40 Kb.(2)Use the rhesus monkey MHC and a cynomolgus monkey MHC from BGI as reference sequences, to identify 298 unique sites, then design recombinant primers and screening primers.After post-testing 79 primers were selected; Ramdom 96 poolings were selected from 312 for low pooling deep sequencing, sequencing data is 2G with sequencing depth of about 2X. Next study is mapping the recombinant primers to the sequencing data, and screening the positive poolings containing MHC sequences.(3)The recombinant cassette was produced by a pair of 75 bp primers, which consisted of 50 bp homology sequence of targeted region and 25 bp AMP amplification primer,then using PCR with Phusion DNA Polymerase and with PMD-18 Tvector as template of AMP gene(1130bp), 135 positive clones were found from the 69 loci with 79 primers with the coverage of 2.8Mb of the MHC sequence.(4) Contigs distributed on MHC display that the contigs were densely distributed in 1080Kb-2200 Kb, 3175Kb-4300 Kb two sections, so we picked out 38 clones which were located in 108Kb-2200 Kb region, fragment and paired-end sequencing of library with 2K,5K insertsize.(5)Gene annotation with the contigs,89 genes non-duplicated were found,which non-classical MHC class I genes were 19, 14 genes belong to MHC III region,and there were 56 genes mapped in human.