目的应用二代测序(next generation sequencing,NGS)技术对CSF1PO和D18S51基因座进行分析检测,研究CSF1PO和D18S51基因座的序列多态性。方法采集165例中国汉族无关个体外周血样,应用QIAamp DNA Mini试剂盒提取样本DNA,Ion Plus Fragment...目的应用二代测序(next generation sequencing,NGS)技术对CSF1PO和D18S51基因座进行分析检测,研究CSF1PO和D18S51基因座的序列多态性。方法采集165例中国汉族无关个体外周血样,应用QIAamp DNA Mini试剂盒提取样本DNA,Ion Plus Fragment Library试剂盒构建文库,在Ion Torrent PGMTM测序平台上进行DNA序列测定,针对新发现的等位基因进行Sanger测序验证。应用Torrent SuiteTMv5.0.2和Integrative Genomics Viewer软件分析数据,进行基因型鉴定和频率统计,运用PowerState v12软件对数据进行统计学处理。结果应用NGS技术同时获得了CSF1PO和D18S51基因座长度多态性和序列多态性,在CSF1PO基因座中,发现了1个新的基因型,在D18S51基因座中,发现2个新的基因型。采用Sanger测序法对NGS技术检测新发现的等位基因进行验证,验证结果一致。结论应用二代测序技术可检测CSF1PO和D18S51基因座核心重复序列的结构,提高基因座的识别效能。展开更多
The custom-designed single nucleotide polymorphism(SNP)panel amplified 231 autosomal SNPs in one PCR reaction and subsequently sequenced with massively parallel sequencing(MPS)technology and Ion Torrent personal genom...The custom-designed single nucleotide polymorphism(SNP)panel amplified 231 autosomal SNPs in one PCR reaction and subsequently sequenced with massively parallel sequencing(MPS)technology and Ion Torrent personal genome machine(PGM).SNPs were chosen from SNPforID,IISNP,HapMap,dbSNP,and related published literatures.Full concordance was obtained between available MPS calling and Sanger sequencing with 9947A and 9948 controls.Ten SNPs(rs4606077,rs334355,rs430046,rs2920816,rs4530059,rs1478829,rs1498553,rs7141285,rs12714757 and rs2189011)with low coverage or heterozygote imbalance should be optimized or excluded from the panel.Sequence data had sufficiently high coverage and gave reliable SNP calling for the remaining 221 loci with the custom MPS-SNP panel.A default DNA input amount of 10 ng per reaction was recommended by Ampliseq technology but sensitivity testing revealed positive results from as little as 1 ng input DNA.Mixture testing with this panel is possible through analysis of the F MAR(frequency of major allele reads)values at most loci with enough high coverage depth and low level of sequencing noise.These results indicate the potential advantage of the custom MPS-SNP assays and Ion Torrent PGM platform for forensic study.展开更多
目的应用Ion Torrent PGM^TM平台对人线粒体基因组全序列进行分析检测。方法采集39名辽宁汉族无关个体以及4个母系家系的14名相关个体样本,应用SequalPrep^TM Long PCR试剂盒进行扩增,应用Ion Shear^TM Plus Reagents试剂盒和Ion Plus ...目的应用Ion Torrent PGM^TM平台对人线粒体基因组全序列进行分析检测。方法采集39名辽宁汉族无关个体以及4个母系家系的14名相关个体样本,应用SequalPrep^TM Long PCR试剂盒进行扩增,应用Ion Shear^TM Plus Reagents试剂盒和Ion Plus Fragment Library试剂盒等构建文库,并在Ion Torrent PGM平台上进行线粒体基因组全序列测序。结果在39名无关个体共观察到39种单倍型,在396个位置观察到了397种碱基变异。无关个体中出现的变异位点数目为25~53个,平均每个个体出现36.2个碱基变异。4个母系家系中每个家系成员间具有完全相同的mtDNA单倍型,严格遵守母系遗传。结论采用本研究建立的人线粒体基因组全序列的测序检验法,可显著提高mtDNA的个体识别能力,在法庭科学领域中有较好的应用价值。展开更多
文摘目的应用二代测序(next generation sequencing,NGS)技术对CSF1PO和D18S51基因座进行分析检测,研究CSF1PO和D18S51基因座的序列多态性。方法采集165例中国汉族无关个体外周血样,应用QIAamp DNA Mini试剂盒提取样本DNA,Ion Plus Fragment Library试剂盒构建文库,在Ion Torrent PGMTM测序平台上进行DNA序列测定,针对新发现的等位基因进行Sanger测序验证。应用Torrent SuiteTMv5.0.2和Integrative Genomics Viewer软件分析数据,进行基因型鉴定和频率统计,运用PowerState v12软件对数据进行统计学处理。结果应用NGS技术同时获得了CSF1PO和D18S51基因座长度多态性和序列多态性,在CSF1PO基因座中,发现了1个新的基因型,在D18S51基因座中,发现2个新的基因型。采用Sanger测序法对NGS技术检测新发现的等位基因进行验证,验证结果一致。结论应用二代测序技术可检测CSF1PO和D18S51基因座核心重复序列的结构,提高基因座的识别效能。
基金supported by grants from the National Natu-ral Science Foundation of China[grant number 81330073],[grant number 81302620]the Ministry of Science and Technology of China[grant number 2016YFC0800703]the Science and Technology Commission of Shanghai Municipality[grant number 14DZ2270800].
文摘The custom-designed single nucleotide polymorphism(SNP)panel amplified 231 autosomal SNPs in one PCR reaction and subsequently sequenced with massively parallel sequencing(MPS)technology and Ion Torrent personal genome machine(PGM).SNPs were chosen from SNPforID,IISNP,HapMap,dbSNP,and related published literatures.Full concordance was obtained between available MPS calling and Sanger sequencing with 9947A and 9948 controls.Ten SNPs(rs4606077,rs334355,rs430046,rs2920816,rs4530059,rs1478829,rs1498553,rs7141285,rs12714757 and rs2189011)with low coverage or heterozygote imbalance should be optimized or excluded from the panel.Sequence data had sufficiently high coverage and gave reliable SNP calling for the remaining 221 loci with the custom MPS-SNP panel.A default DNA input amount of 10 ng per reaction was recommended by Ampliseq technology but sensitivity testing revealed positive results from as little as 1 ng input DNA.Mixture testing with this panel is possible through analysis of the F MAR(frequency of major allele reads)values at most loci with enough high coverage depth and low level of sequencing noise.These results indicate the potential advantage of the custom MPS-SNP assays and Ion Torrent PGM platform for forensic study.
文摘目的应用Ion Torrent PGM^TM平台对人线粒体基因组全序列进行分析检测。方法采集39名辽宁汉族无关个体以及4个母系家系的14名相关个体样本,应用SequalPrep^TM Long PCR试剂盒进行扩增,应用Ion Shear^TM Plus Reagents试剂盒和Ion Plus Fragment Library试剂盒等构建文库,并在Ion Torrent PGM平台上进行线粒体基因组全序列测序。结果在39名无关个体共观察到39种单倍型,在396个位置观察到了397种碱基变异。无关个体中出现的变异位点数目为25~53个,平均每个个体出现36.2个碱基变异。4个母系家系中每个家系成员间具有完全相同的mtDNA单倍型,严格遵守母系遗传。结论采用本研究建立的人线粒体基因组全序列的测序检验法,可显著提高mtDNA的个体识别能力,在法庭科学领域中有较好的应用价值。
