Application Of Next-generation Sequencing On Study Genomic Structural Variants

Copy Number Variants(CNVs)is a common type of genomic structural variants,and chromosomal microarray analysis is now widely accepted as the best tool for detecting CNVs.Patients with neurofibromatosis 1(NF1)has the only clinical manifestation of cafe-au-lait macules at very early stage.For qualify the diagnosis criteria for NF1,patients need to develope a second clinical phenotype after these macules.With the help of sequencing and microarray platform,we can detect single neucleotide variants,small indels and CNVs at the same time.With these genetic information we can diagnose NF1 patients early without the second phenotype and could provide information for clincal conseling.In this study,we have diagnosed 13 patients in which we found 4 recurrent stop condon mutations and 9 novel mutations including stop condon mutation,splicing mutation,frame-shift mutation and large deletions.Thus,applying genetic testing in NF1 patients could help to improve the diagnostic efficency.And we observed in our study that typical macules with regular borad and color are more related with NF1 patients.Whole Exome Sequencing(WES)is now used clinically for diagnosis of genetic diseases as the quality of WES has improved greatly.Though the first choice for detecting CNVs is microarray,we are still interested in those algorithms using WES data as input based on read depth method.In this study we evaluted three different algorithms(XHMM,CoNIFER and CNVnator)using CNVs detected by microarry as standard.The output of WES based algorithms consists of smaller vairants less than 50 Kb.Sensitivity of XHMM and CoNIFER are not satisfying for vairants with less probes.CNVnator has higher sensitivity in the cost of high false positive rate,and CNVnator are more accurate with larger CNVs.Breakpoint of structual variants is always an important part in study of human genome.Previous method for interogating structual variants is laborious and suffers from low resolustion.We have established a capturing method called breakpoint spreading anchored PCR,which can be used to capture targeted region for sequencing without prior information of the other side of the breakpoint.We has successfuly mapped two breakpoints in patients,one with balanced translocation and one with interspersed duplication.Thus Breakpoint Spreading Achored PCR is an efficient way to capture and precisely mapping breakpoint of structual variants.