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想测SSR,想到创新技术—SSRseq
摘要: SSR检测新方法介绍
         新技术方法的产生发展从来都是来势汹汹、锐不可当的,SSR的检测亦是如此!自从生命科学迈入到ATCG时代以来,传统的仅仅靠“跑电泳,比大小”的定性分析,已经越来越无法满足科研工作者对“准确、高效、定量”的要求了。二代高通量测序技术的发展,让这一目标成为现实,天昊生物创新技术—SSRseq,把SSR的“序列信息”及“比例信息”一网打尽!下面就跟随小编看看SSR检测的“前世今生”。
 
什么是SSR?

        SSR (Simple Sequence Repeats,简单序列重复),或称STR(Short Tandem Repeat,短片段串联重复)或者Microsatellites(微卫星),广泛的存在于真核生物基因组中。大多数SSRs是非编码序列,可以影响基因表达、剪接、蛋白序列及基因组结构等(1-5)。SSR长度突变频率在每一世代每个位点大概是10-7到10-3之间(6),这远远高于单个碱基10-9左右的突变频率(7-8),从而在基因组中产生了更具多样性的SSRs。尽管SSR序列自身具有高度的变异性,但是它侧翼区域的序列却在物种内具有很高保守性,有时这种保守型甚至在物种间存在(9-12)。SSR相较与其他遗传变异具有几方面特点,包括共显性、高度可重复性和DNA检测需要量少等(13-16)。更重要的是,这种SSR序列的多样性和它侧翼序列保守性的结合,使它成为一种理想的遗传分子标记。的确,SSRs已经在包括DNA指纹图谱分析、基因作图、亲缘关系鉴定、分子辅助育种、遗传多样性分析、种子纯度及品系鉴定中发挥着重要的作用(16-20)。
 
SSR多样性的产生原因及传统检测的不足
        SSR多样性产生的最主要原因是在SSR复制过程中DNA聚合酶固有的“滑移”现象(Slippage)造成的(21-27),这种滑移现象同样可以发生在体外,导致错误的SSR等位基因并增加了SSR准确分型的难度。而且,基于琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳、毛细管电泳这些目前常用的SSR检测方法,普遍存在着分辨率不高、不够准确、效率及通量不高等问题。例如,目前在冬菇、黄麻和木豆中进行的DNA指纹图谱分析仅仅用到25、28和48个SSR位点(28-30)。这些有限的SSRs不足以构建高质量SSR指纹图谱用于区分亲缘性高物种间的关系。全基因组重测序虽然一次可以检测大量SSR位点(25, 31-32),但是SSR序列仅仅占整个基因组的很小部分,例如人类基因组中的SSR只占3%左右(33),因此全基因组重测序会获得很多我们关心的SSR以外的冗余序列,这就稀释了所测有用数据的比例,使得SSR位点的测序深度很难超过10-100X(25),这样在合理的测序价格内,利用全基因组重测序的方法就难以得到准确性高的SSR分型。另外,用全基因组重测序进行SSR分型还会导致某些SSR位点的扩增偏好性以及SSR重复序列较高难度的数据分析等问题(34-36)。
 
       天昊生物自主研发的基于二代测序技术的SSR分型新方法--SSRseq,这种方法几乎克服了现存所有检测方法的不足,尤其适合对多SSR位点、超高深度的分型,准确度高,并且分辨率达到单碱基的水平。因此适合所有二倍体动植物及真核微生物的SSR位点分型。另外,我们还成功对六倍体植物—油茶进行了SSR分型。对于多倍体物种来说,我们的SSRseq可以提供不同等位基因的比例数据,从而提高了多倍体物种遗传多样性分析的准确度,获得更加清晰的遗传结构图。
 
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部分内容来源:doi: 10.1093/nar/gkx093
 
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