第二代测序教材.ppt

* * This is the result of a reset: the extended template strand is melted off and the sequencing template re-exposed as a fresh, clean template and devoid of any noise generated by previous sequencing cycles. In this method, noise is generated by the attrition of strands at each cycle (uncompleted extensions) that serve to reduce template number (and therefore, fluorescence intensity) on each bead, and increases the noise-to-signal ratio of each bead. The ability to ‘reset’ is one of the major benefits of this chemistry. A major problem that limits read length of all NGS systems is that as the read gets longer, the signal falls and noise rises, until you can no longer accurately call a base. By resetting the system every 5 cycles we remove all the accumulated noise at each sequencing round. * * 变性洗脱。进行下一轮测序 * * * 双碱基编码矩阵，一个荧光信号不能知道什么碱基，必须已知一个碱基。 0位就是测序引物5端的第一个碱基。 * * This slide explains how to decode a sequence. All you need to decode a base sequence is to know IN this instance we will say we know the first base is an A [important it does not need to be the first base as long as you are certain of one of the bases then the decoding is automatic] Remember that the 2nd base in each decoded pair is the first base of the next pair In this example we know the first base is an A using the lookup table 1st base A and blue color tells us second base is an A Now moving to the second observed color we know the first base this pair must be an A so if we see 1st base A and green signal 2nd base must be a C and so on …………………………………………. 待测模板结合到带有P1引物的磁珠上，乳液pcr的扩增过程，基于DNA连接酶，不是通常我们用的DNA聚合酶 片段，配对末端 大家知道生物体内的基因组DNA包含着几乎全部的生物遗传信息，因此要想透彻了解生物体的整个生命过程的本质，我们必须获得该生物体的整个DNA序列信息，因此测序技术的进步从某种意义上来说，决定了整个生物学研究的水平和深度。 测序技术可以追溯的20世纪的50年代，早在1954 化学降解方法，操作复杂，并没有被广泛使用。 1977 双脱氧核苷酸末端终止法，一直在使用 1977 化学降解方法，原理同sanger，加入某种化学试剂，对DNTP的5‘端的磷酸基团进行标记，化学修饰，裂解DNA片段，形成一系列大小不一的小分子DNA片段，电泳 80年代中期，荧光标记，代替同位素标记，自动荧光信号接收器，计算机分析系统，代替放射性自显影。 1996毛细管电泳，提高通量。 两个比较核心的专利技术 1、生成DNA簇的过程 2、可逆性的末端终止 反应体系中加入带有四色