想測SSR，想到創(chuàng)新技術(shù)—SSRseq

發(fā)稿時間：2017-08-11來源：天昊生物

摘要：SSR檢測新方法介紹

新技術(shù)方法的產(chǎn)生發(fā)展從來都是來勢洶洶、銳不可當(dāng)?shù)?，SSR的檢測亦是如此！自從生命科學(xué)邁入到ATCG時代以來，傳統(tǒng)的僅僅靠“跑電泳，比大小”的定性分析，已經(jīng)越來越無法滿足科研工作者對“準(zhǔn)確、高效、定量”的要求了。二代高通量測序技術(shù)的發(fā)展，讓這一目標(biāo)成為現(xiàn)實，天昊生物創(chuàng)新技術(shù)—SSRseq，把SSR的“序列信息”及“比例信息”一網(wǎng)打盡！下面就跟隨小編看看SSR檢測的“前世今生”。

什么是SSR？

SSR (Simple Sequence Repeats，簡單序列重復(fù))，或稱STR（Short Tandem Repeat，短片段串聯(lián)重復(fù)）或者Microsatellites（微衛(wèi)星），廣泛的存在于真核生物基因組中。大多數(shù)SSRs是非編碼序列，可以影響基因表達、剪接、蛋白序列及基因組結(jié)構(gòu)等（1-5）。SSR長度突變頻率在每一世代每個位點大概是10^-7到10^-3之間（6），這遠遠高于單個堿基10^-9左右的突變頻率（7-8），從而在基因組中產(chǎn)生了更具多樣性的SSRs。盡管SSR序列自身具有高度的變異性，但是它側(cè)翼區(qū)域的序列卻在物種內(nèi)具有很高保守性，有時這種保守型甚至在物種間存在（9-12）。SSR相較與其他遺傳變異具有幾方面特點，包括共顯性、高度可重復(fù)性和DNA檢測需要量少等（13-16）。更重要的是，這種SSR序列的多樣性和它側(cè)翼序列保守性的結(jié)合，使它成為一種理想的遺傳分子標(biāo)記。的確，SSRs已經(jīng)在包括DNA指紋圖譜分析、基因作圖、親緣關(guān)系鑒定、分子輔助育種、遺傳多樣性分析、種子純度及品系鑒定中發(fā)揮著重要的作用（16-20）。

SSR多樣性的產(chǎn)生原因及傳統(tǒng)檢測的不足

SSR多樣性產(chǎn)生的最主要原因是在SSR復(fù)制過程中DNA聚合酶固有的“滑移”現(xiàn)象（Slippage）造成的(21-27)，這種滑移現(xiàn)象同樣可以發(fā)生在體外，導(dǎo)致錯誤的SSR等位基因并增加了SSR準(zhǔn)確分型的難度。而且，基于瓊脂糖凝膠電泳、聚丙烯酰胺凝膠電泳、毛細管電泳這些目前常用的SSR檢測方法，普遍存在著分辨率不高、不夠準(zhǔn)確、效率及通量不高等問題。例如，目前在冬菇、黃麻和木豆中進行的DNA指紋圖譜分析僅僅用到25、28和48個SSR位點（28-30）。這些有限的SSRs不足以構(gòu)建高質(zhì)量SSR指紋圖譜用于區(qū)分親緣性高物種間的關(guān)系。全基因組重測序雖然一次可以檢測大量SSR位點（25, 31-32），但是SSR序列僅僅占整個基因組的很小部分，例如人類基因組中的SSR只占3%左右（33），因此全基因組重測序會獲得很多我們關(guān)心的SSR以外的冗余序列，這就稀釋了所測有用數(shù)據(jù)的比例，使得SSR位點的測序深度很難超過10-100X（25），這樣在合理的測序價格內(nèi)，利用全基因組重測序的方法就難以得到準(zhǔn)確性高的SSR分型。另外，用全基因組重測序進行SSR分型還會導(dǎo)致某些SSR位點的擴增偏好性以及SSR重復(fù)序列較高難度的數(shù)據(jù)分析等問題（34-36）。

天昊生物自主研發(fā)的基于二代測序技術(shù)的SSR分型新方法--SSRseq，這種方法幾乎克服了現(xiàn)存所有檢測方法的不足，尤其適合對多SSR位點、超高深度的分型，準(zhǔn)確度高，并且分辨率達到單堿基的水平。因此適合所有二倍體動植物及真核微生物的SSR位點分型。另外，我們還成功對六倍體植物—油茶進行了SSR分型。對于多倍體物種來說，我們的SSRseq可以提供不同等位基因的比例數(shù)據(jù)，從而提高了多倍體物種遺傳多樣性分析的準(zhǔn)確度，獲得更加清晰的遺傳結(jié)構(gòu)圖。

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部分內(nèi)容來源：doi: 10.1093/nar/gkx093

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