Sequence Recognition And Hotspot Selective Preference Research Of Chimeras And Its Application Exploration In Haplotype Analysis

In recent years,Whole genome amplification(WGA)based on multiple displacement amplification(MDA)has been extensively applied in many recent genomic researches because of the higher amplification efficiency and higher fidelity and isothermal amplification of the phi29 DNA polymerase.However,the phi29 DNA polymerase will produce a certain number chimeric sequences(i.e.chimeras)during the process of phi29 multiple displacement amplification(i.e.phi29MDA).In this research,we combined the previous study of chimeras,designed and established a set of simple and highly efficient chimera recognition pipeline based on BWA alignment software,which classified and analyzed numerical proportion of the recognized chimeric sequences.Meanwhile,we devised process for scanning the human genome sequence and seeking the chimeric hotspot areas which easy to generate chimeras.Moreover,we explored chimeric hotspots selective preference of chimeras.According to the previous research,the two parts of chimeras come from the same template and their genome distance could be over 5000nt.This phenomenon indicated that chimeras can be used in haplotype assembly.Our results of this research include:(1)A bioinformatics pipeline based on BWA alignment software was designed to recognize chimeras from alignment result of sequence data and realized the classification and quantity statistics of chimeras.In the same sequence data from phi29MDA,compared our pipeline with pre-existing chimeras’ analysis pipeline,new pipeline can clearly recognize chimeras’ category and have more efficient and consume less time.(2)Based on the overlap fragment and sequence distance distribution between chimera fragments,we described the characteristics of the potential generation region of chimeric sequence in the genome and constructed a scanning pipeline for searching the chimeric hotspot areas which can easy to form chimeras in the human genome.The results of scanned chimeric hotspots indicated that chimeric hotspots with different length of overlap fragments present random distribution in the human genome,the relationship between the number of chimeric hotspots and the length of chromosome is linear.(3)Conjoint analyzed chimeras of MDA sequence data and chimeric hotspots of the human genome,we found that there is no chromosome selective preference of chimeras,distance of chimera’s fragments presents the peak over 80-280nt.Through comprehensive the selective preference of chimeras on length and GC content of overlap fragments,we know that chimeras are more likely to generate from the chimeric hotspot areas which the denaturation temperatures(i.e.Td)of overlap fragments is similar with the reaction temperature of MDA.This conclusion is conducive to optimizing MDA reaction system and reduce the chimeras.