MMM第二代测序技术剖析.ppt

* Long Reads in the curve format. 1260 base read by John Gardner at CEPRAP, Davis, CA * * * 64 lanes can be loaded in 5 minutes with the 8-channel Hamilton syringe. The syringe is compatible with microtiter plates. The syringe will load every 4th lane. For sequencing, each half of the gel is loaded separately (first set of 8 reactions) . On the first half the A lanes are loaded, then the C lanes etc. The same procedure is performed on the second half of the gel. * * 1）样品输入并片段化 2）序列两端接头连接，文库制备3）磁珠吸附，乳化PCR扩增 测序流程依次加入五种引物进行五次测序。加入测序引物及加入oligo连接酶连接，激发荧光检测，循环一个流程，换一种引物再循环一个流程，五个流程结果叠加分析出序列。 Paired-End方法，基因组打断后，选择一定长度（200－500bp)的序列连接两端接头进行两头测序。Mate-end建库较复杂，序列打断后，选取一定长度序列(3-5kb)，需先连接生物素，再环化，再打断，生物素富集，连接两端接头进行两端测序。 Hom error rate纯合子SNP错误率，HET error rate杂合子SNP错误率。 a, Power calculations showing the number of true somatic substitutions detected (blue) and mis-calls (single nucleotide polymorphisms (SNPs) called as somatic mutations, burgundy, and sequencing errors called as mutations, green) for different levels of sequence coverage. Calculations are based on a true mutation prevalence of 1 per megabase (black line).?b, Histogram of the actual coverage achieved per base of the tumour (blue) and normal (burgundy) genomes.?c, Figurative representation of the catalogue of somatic mutations in the genome of NCI-H209. Chromosome ideograms are shown around the outer ring and are oriented pter–qter in a clockwise direction with centromeres indicated in red. Other tracks contain somatic alterations (from outside to inside): validated insertions (light-green rectangles); validated deletions (dark-green rectangles); heterozygous (light-orange bars) and homozygous (dark-orange bars) substitutions shown by density per 10 megabases; coding substitutions (coloured squares; silent in grey, mis-sense in purple, nonsense in red and splice site in black); copy number (blue lines); validated intrachromosomal rearrangements (green lines); and validated interchromosomal rearrangements