高通量測序技術的原理及各平臺優勢和實踐應用的分析6

應用

WGS正在成為NGS中最廣泛的應用。通過該技術并且結合生物學應用，研究人員可以獲得基因組信息中最值得注意的信息73。舉例來說，2012年，Ellis等報道了基因與乳腺癌患者芳香酶抑制劑（aromatase inhibitor）治療法之間的關聯。他們指出突變，后果與診斷之間的關聯，同樣還有癌癥相關基因的突變的富集。這提供了一個可能性，即：乳腺癌有不同的突變造成不同的表型，具有復雜的病理學75。最近的NGS平臺的改進使得研究人員發現了一些幾年前難以想象的新觀點與機會。在2010年，1000基因組計劃（1000 genomes project）開放了其從179個個體中獲得的WGS原始數據以及697個個體的測序數據76。到2015年，研究人員已經構建了26個不同人群的2504個人的基因組群體77,78。給人們從種群的角度來觀察人類的變異。但這還不是該項目的終點，越來越多的人的基因組正在被得以測序79-81。種群水平的測序已經成為人們更好的理解人類疾病的一個重要的工具，同樣也得到了意想不到的結果。一個例子是，Sidore等82對2120個撒丁島人（Sardinians）的WGS研究發現了一些新的和脂肪相關的基因以及炎癥的標志物，給人們對血液膽固醇的分子機制的研究提供了新思路。

全外顯子組測序（Whole-exome and targeted sequencing）83同樣也廣泛應用于測序的研究中。受制于基因組材料大小的局限，很更多的個人樣本可以在一個測序中實現，增加了基因組研究的寬度以及深度。使用外顯子測序，Iossifov84等對超過2500個單一的家庭進行測序，每個家庭都有一個小孩患有自閉癥（autism spectrum disorder, ASD）。研究人員在30%的樣本中發現了錯意突變（missense mutations），基因干擾的突變（gene-disrupting mutations）以及拷貝數的變異。該工作與其他的工作一道鑒定到了ASD相關的基因突變85,86。其他證據表明，高覆蓋度的WGS也可以解決復雜的變異以及臨床樣本的分析。2015年，Griffith等認為可以使用一個完美的跨平臺的方法（包含靶向測序）來鑒定腫瘤中高可信度的SNPs。該方法中，作者認為10000×的覆蓋度可以鑒定到稀有突變。由于10000×的覆蓋度對于WGS而言實在過高，靶向測序便在臨床中得到了廣泛的應用。

 NGS同樣在基因的調控研究中有廣泛的應用。蛋白-DNA互作可以通過染色質免疫共沉淀結合NGS測序（ChIP-seq）來得以研究41。利用NGS對修飾堿基的研究也是可行的。舉例來說，甲基化測序包含了甲基化DNA的捕獲與富集88，對甲基化與非甲基化區段的選擇性消化89,90,91。但是，盡管利用此方法獲得了很多重大的發現，修飾與捕獲過程成為其最大的限制。2010年，Flusberg等92發表了一個概念性的研究方法，即：使用PacBio來區分甲基化與非甲基化的堿基。由于聚合酶即便是甲基化的堿基也能夠延伸，但在甲基化位點上會停留更多的時間，因此這里改變的信號可以認為含有甲基化修飾。與之相同的是，nanopore平臺也能夠監測修飾的堿基，因為甲基化同樣會影響鑒定到的電壓的變化。這使得甲基化的測序可以在不需要化學操作的條件下進行93。

一個最近的NGS的范例是對長鏈DNA的測序。重復序列以及復合序列長久以來較難以拼接，短讀長測序很難解決這個問題94-96。最近，Chaisson等97對長讀長測序的使用使得其能夠在人類GRCh37數據庫中提交超過1Mb的新的序列，這些序列彌補甚至跨越了曾經的溝。Chaisson等還鑒定到了大于26000個超過50bp的indels，也因此，GRCh37數據庫成為最有參考價值的幾個基因組之一。除了簡單的增加基因組數據可靠性之外，長讀長還能夠提供更有效的臨床診斷98-100。

在對轉錄水平上的研究也因為NGS受益匪淺。今天，研究人員甚至能夠使用NGS的深度測序對單個轉錄本進行研究。2014年，Treutlein等101使用了組織發育過程中不同細胞類群的單細胞RNA測序發現了用于鑒定細胞亞群的標志物。盡管長讀長測序相對而言在對轉錄本的定量上不占優勢，但是，長讀長可以在研究轉錄組的結構上有所幫助51。舉例來說，最近的人類長讀長轉錄組測序研究表明 >10%的reads是新的可變剪切體102。

 NGS最新的設備——nanopore測序儀，依然在尋找其定位的過程中。然而，研究人員正在將其快速的文庫制備，實時的數據生產以及小的體積的優勢轉變為資本過程中。最近，英國Stanley Royd Hospital的研究人員使用MinION用于監測沙門氏菌（Salmonella enterica）的爆發103。MinION測序儀最令人振奮的應用可能就是2014年的埃博拉病毒爆發104。在位于日內瓦的歐洲移動實驗室的主持下，作者對埃博拉病毒的傳播以及進化歷史進行了深入的研究。

結尾
我們正處在新的NGS技術革命的頂端。NGS現在已經不僅僅只是一個新奇的事物，而已經成為了一個在生物學研究中廣泛應用的技術。最新的超高通量測序儀已經將曾經認為不可能的事情成為可能。這包含了首創的精準醫療（medicine initiatives）以及Illumina計劃的對循環腫瘤DNA（circulating tumour DNA, ctDNA）進行測序。每個計劃都對數萬個基因組樣本進行測序。所以，快速以及低成本的測序給予了內科醫生強大的工具來翻譯基因組信息成為有用的臨床診斷結果。

這個革命也帶來了新的挑戰。由于NGS旨在廣泛的應用于臨床，時間就成為一個NGS首先需要面對的挑戰。對于那些嚴重的神經性疾病或者極為危險的癌癥患者而言，數周的WGS分析的等待時間足以使的患者錯過最佳的治療時間。對于急性感染而言，這些事件已經下降到幾天。盡管人們已經對時間做出了巨大的改進，但是絕大多數現有的系統都不能完全滿足快速模式下的足夠產出。

雖然臨床診斷面臨著數據量不夠的問題，NGS其他方面的應用卻面臨著生產力過剩的境地。目前，已有超過14000個基因組序列上傳到US National Center for Biotechnology Information（NCBI）中。2013年，Schatz與Langmead報道了全世界每年可以生產超過15pb的數據量，并且數量與通量依然在繼續增加107。數據量的富余對分析以及其下游提出了嚴峻的挑戰，這需要革命性的存儲與生信解決方案108。將海量的數據量翻譯成有生物學與遺傳學內涵的結果同樣也是一個挑戰87,109,110。在臨床診斷方面，通過NGS分析的數據產生的假陽性或者假陰性同樣也是需要慎重考慮的問題111,112。

最近，Illumina由于NGS與其周邊產品獲得了巨大的成功。其它生產商也在快速革新自身的產品113。Illumina的市場仍然在增長，以至于優勢巨大。BGISEQ-500以及Helicos technology的GenoCare114在亞洲也有所斬獲。ONT PromethION115與Illumina HiSeq X系列則向著成本與產量的極限大步邁進。隨著人們對臨床診斷測序興趣的增加，已有的NGS供應商正在提供各種快速的解決方案，如Ion Torrent S5以及Illumina的MiniSeq，還有新加入者Qiagen的GeneReader也來參與競爭。

今后的幾年里，更多的玩家也會帶著心得解決方案進入這個市場。GenapSys (Sigma-Aldrich)的electronic ‘lunchbox’-sized sequencer116; Genia (Roche)的新的nanopore測序方案117; 以及單通道CMOS技術118，都號稱能夠在臨床應用上節約足夠的時間。這些已有的和新的攪局者都有著科技革命的潛質，包括直接對RNA或者蛋白進行測序等，這些最近和未來的進步使得今天成為NGS發展的黃金時期。

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