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一种基于新冠病毒的全基因组全长扩增的三代建库测序方法

2021-02-20 04:02:01

一种基于新冠病毒的全基因组全长扩增的三代建库测序方法

　　技术领域

　　本发明涉及基因测序技术领域，尤其涉及一种基于新冠病毒的全基因组全长扩增的三代建库测序方法。

　　背景技术

　　新型冠状病毒是一种具有包膜的、不分节段的正链单股RNA病毒，颗粒呈圆形或椭圆形，直径约80～120nm，属于网巢病毒目冠状病毒科Betacoronavirus。病毒粒子被宿主细胞提供的脂质双层所包裹，其中含有核酸及核衣壳蛋白，有三种主要蛋白：包膜蛋白(E蛋白)、膜蛋白(M蛋白)和刺突蛋白(S蛋白)。此病毒每组基因组长度约三万个核苷酸左右，基因序列显示SARS-CoV-2属于乙型冠状病毒属谱系β(Betacoronavirus Lineageβ,Sarbecovirus)进化树中分支较长的一种病毒，与中华菊头蝠中发现的冠状病毒相似，例如MERS-CoV或SARS-CoV。对病毒的生物遗传学分析显示，同属人类冠状病毒的SARS病毒分离株AY274119相比MERS病毒分离株KC164505、JX869059等在亲缘关系上距离SARS-CoV-2病毒更近。

　　2019-2020年，新型冠状病毒肺炎在全球疯狂肆虐，病毒潜伏期平均大约3-7天，最长不超过14天。大多数患者的表现以下呼吸道症状为主，常见临床表现包括发热、四肢乏力、干咳等症状，其他表现包含鼻塞、流鼻涕、头痛、咽痛、咳血，咳痰、或腹泻等。有部分患者仅表现为低热、轻微乏力等，无肺炎表现。还有部分患者无任何临床表现。病毒感染严重后，会引发多种并发症包含急性呼吸窘迫综合征(ARDS)、脓毒症休克、全身炎症反应综合征、难以纠正的代谢性酸中毒、急性心肌损伤，和出凝血功能障碍等。

　　针对新型冠状病毒引发的疫情，目前确诊主要是基于荧光PCR的技术，利用引物探针去扩增病毒靶序列，根据目前中国疾控中心出的诊疗方案中提供的三个靶点，有任何两个靶点呈现阳性，都可以断定病毒为新型冠状病毒2019-nCov。检测结果若呈阳性就会呈现出一条曲线；阴性就是一条平线。

　　除荧光PCR检测技术外，基于检测样本的宏基因组测序(Meta)、探针捕获测序(Capture)和多重PCR扩增子测序(Amplicon)等方法可以提供更加精确的检测。扩增子(amplicon)为DNA或RNA扩增后的一段核苷酸序列。比如通过PCR扩增得到的某个基因的扩增片段。扩增子测序是一种高靶向性方法，用于分析特定基因组区域中的基因变异。已有的新冠病毒扩增子技术都是针对病毒的短片段扩增与二代测序，而基于三代测序平台PacBio的病毒全基因组全长扩增子测序可轻松读取病毒的全长序列，突破了二代测序读长较短的局限性，提高了病毒的分辨能力。病毒全基因组扩增测序获得全部变异区域序列信息极大的提高了物种鉴定的分辨率与准确率。

　　然而目前针对新冠病毒基因序列PCR扩增技术准确率有待提高，PacBio三代测序的长读长优势并没有完全发挥，尚未有研究根据新冠病毒的基因组序列设计引物达到可覆盖整个病毒全基因组的简单扩增技术，也未有研究基于PacBio三代测序获得99％以上的长读长序列，用于病毒的变异检测、组装、溯源分析等。

　　发明内容

　　本发明针对病毒的全基因组序列，结合扩增子测序的技术特点与PacBio HiFiReads的测序优势，针对PacBio测序平台开发出了一套简单快速的新冠病毒全长扩增的建库测序方法。

　　为了达到上述目的，本发明提出了一种基于新冠病毒的全基因组全长扩增的三代建库测序方法，包括以下步骤：

　　1.引物的设计与合成

　　1)获取多条新冠病毒序列，处理、比对后获得一致性序列，其核苷酸序列如SEQ IDNO.1所示；

　　2)基于一致性序列设计引物，引物覆盖整个新冠病毒基因组；

　　进一步地，所述引物位置基于基因组的保守区域设计；

　　进一步地，所述引物包含30条奇数引物和30条偶数引物，引物的核苷酸序列如SEQID NO.2～SEQ ID NO.61所示；

　　进一步地，每对引物之间具有约100bp的overlap；

　　进一步地，每对引物扩增的PCR产物大小为0.9～1.2k。

　　2.新冠病毒基因组全长扩增

　　1)配制扩增反应，每个样本分别使用奇数引物混池与偶数引物混池进行反应的配制，配制完成混匀并进行离心，进行PCR扩增；

　　进一步地，所述扩增反应配液为：新冠病毒cDNA样本，引物混池(奇数或偶数)，KAPAHiFi高保真缓冲液，KAPAdNTP混合液，KAPAHiFi酶，无核酸酶水；

　　进一步地，所述PCR扩增条件为：98℃保持2min；98℃保持20s，65℃保持30s，72℃保持1.5min，共35个循环；72℃保持5min。

　　2)进行磁珠纯化，洗脱。

　　3)取纯化产物，稀释，进行Qubit定量，确定PCR产物的浓度与总量；取稀释液进行凝胶电泳检测检测，确定扩增片段的大小分布。

　　3.全基因组扩增子样本混样：根据Qubit定量结果，将同一样本的奇数扩增产物与偶数扩增产物进行等量混样；

　　进一步地，所述扩增产物混样后的总量在1ug以上。

　　4.文库构建

　　1)末端修复：取全基因组扩增子样本，与反应体系充分混匀，瞬时离心，孵育；反应完成后，进行磁珠纯化，去除反应时加入的酶与Buffer，洗脱，获得片段末端加A的粘性末端；

　　进一步地，所述反应体系为：全基因组扩增子样本，DNA Damage Repair Buffer，End Prep Enzyme。

　　2)连接测序接头：在连接酶作用下，进行连接反应，连接带barcode的测序接头，然后消化外切酶，纯化；

　　进一步地，所述连接反应体系如下：取修复纯化产物，Rapid Ligation Buffer，Rapid DNA Ligase，BarcodeAdapter，混匀，瞬时离心，孵育后，置于冰上；

　　进一步地，所述消化外切酶步骤反应体系如下：取连接产物，外切酶III，外切酶VII，混匀，瞬时离心，孵育置于冰上。

　　5.三代测序：文库质检合格后，用三代测序平台测序。

　　进一步地，所述文库质检方法为，取文库进行Qubit定量，获得文库浓度；进行的片段大小分析；

　　进一步地，所述三代测序平台为PacBio Sequel II测序平台。

　　与现有技术相比，本发明的有益效果为：

　　(1)本专利的方法基于PCR扩增，在样本总要求上非常低，样本量达到1ng以上即可进行扩增后测序。

　　(2)本方法针对新冠病毒的基因组一致性序列，设计了可覆盖整个病毒全基因组的扩增引物，扩增片段大小在1.1k，可以充分发挥PacBio三代测序的长读长优势，同时准确率也可以达到99％以上，可以对新冠病毒进行组装与单倍型分析等。

　　(3)本方法通过将奇数引物与偶数引物混合为混池的方法进行扩增，将需要30个单独的扩增反应简化为仅需要2个扩增反应，极大的简化了实验操作，提高了实验效率，同时，奇数引物和偶数引物混池内部的片段间没有重叠部分，可以防止产生扩增偏差，同时奇数引物与偶数引物扩增片段相互补充，混合后即可覆盖整个新冠病毒的全基因组。

　　(4)本方法通过扩增，可以将测序数据中的病毒数据比例提高几百倍至几十万倍，可以对病毒信息进行富集，极大的降低了测序数据的浪费。

　　附图说明

　　图1为新冠病毒引物位置示意图。

　　图2为实验方案流程示意图。

　　图3为奇数引物与偶数引物扩增检测峰图。

　　图4为文库检测峰图。

　　具体实施方式

　　下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。

　　实施例1：

　　1.引物设计与合成

　　在NCBI上搜索新冠病毒，共下载获得74条新冠病毒序列，通过检查序列完整性，删除长度在20k以下的序列，共获得了43条完整的新冠病毒序列。所有43条完整基因组序列经比对后获得一致性序列，新冠病毒基因组一致性序列如SEQ ID NO：1所示。

　　基于一致性序列，共设计30对引物，引物覆盖整个新冠病毒基因组，引物位置基于基因组的保守区域设计，每对引物之间具有约100bp的overlap，每对引物扩增的PCR产物大小0.9～1.2k。引物设计位置如图1所示，本方案的引物共包括30对，每对引物的扩增片段大小为0.9～1.2k，设计在新冠病毒的保守序列位置。在图中，第一个片段的F端引物标注为1F，R端引物标注为1R，相邻片段间具有约100bp的重叠部分，引物覆盖整个病毒的全基因组区域。在进行扩增时，为确保不会出现小片段干扰，简化扩增操作流程，将引物混合为两个引物池，即将引物名称标注为1、3、5等奇数的引物混合为奇数引物池，将引物名称标注为2、4、6等偶数的引物标注为偶数引物池，每个引物池共包括30条引物序列，新冠病毒样本经过两个引物池的扩增后，即可获得覆盖整个病毒基因组的全长序列。如果奇数和偶数引物不分开，即便分作两个引物池，各含30条引物序列，单个引物池扩增后会优先扩增重叠部分的小片段，出现干扰。

　　设计的引物序列如表1所示：

　　表1：基于一致性序列设计的30对引物

　　2.新冠病毒基因组全长扩增

　　设计的引物合成于上海生工，将引物统一稀释至10uM，将奇数名称编号的引物等体积混合为奇数引物混池，将偶数名称编号的引物等体积混合为偶数引物混池。针对新冠病毒cDNA样本，使用奇数引物混池与偶数引物混池分别进行扩增反应，一份2个新冠病毒cDNA样本的项目实施例操作如下：

　　1)配制扩增反应，条件如表2：

　　表2：

　　每个样本分别使用奇数引物混池与偶数引物混池进行反应的配制，配制完成混匀并进行离心，按照如下的PCR条件进行PCR扩增：98℃保持2min；98℃20s，65℃30s，72℃1.5min，共35个循环；72℃保持5min。

　　2)反应完成后，按照AMPure磁珠的使用说明进行磁珠纯化，最后使用12μL洗脱缓冲液洗脱；

　　3)取1μL纯化产物，使用无核酸酶水稀释5倍，取2μL稀释液进行Qubit定量，确定PCR产物的浓度与总量；取3μL稀释液进行凝胶电泳检测检测，确定扩增片段的大小分布，正常的扩增会在1.1k处看到清晰明亮的条带，如图3所示。

　　3.全基因组扩增子样本混样

　　根据Qubit定量结果，将同一样本的奇数扩增产物与偶数扩增产物进行等量混样，要求混样后的总量在1ug以上。

　　4.文库构建

　　1)末端修复。取1ug的全基因组扩增子样本，进行末端修复反应体系的配制，配制如表3条件反应。

　　表3：

　　充分混匀，瞬时离心，20℃孵育30min。

　　反应完成后，按照AMPure磁珠的使用说明进行1X磁珠纯化，去除反应时加入的酶与Buffer，最后使用14μL洗脱缓冲液洗脱，获得片段末端加A的粘性末端。

　　2)连接带barcode的测序接头

　　通过末端修复与加A后，在加入具有与A末端匹配的带barcode的测序接头，在连接酶的作用下即可实现接头的连接。同时，由于酶切与连接反应存在一定的效率问题，需要使用外切酶III和外切酶VII将结构不完整的文库消化掉。具体实施例的操作流程如下：

　　进行连接反应，反应体系如表4。

　　表4：

　　混匀，瞬时离心，20℃孵育60min，反应结束后65℃孵育10min，置于冰上。进行外切酶消化，反应体系如表5。

　　表5：

　　混匀，瞬时离心，37℃孵育60min，置于冰上。按照AMPure磁珠的使用说明进行磁珠纯化，最后使用20μL洗脱缓冲液洗脱，获得的适用于PacBio测序平台的哑铃型的环状文库。

　　5.文库质检与上机测序

　　取1μL文库进行Qubit定量，获得文库浓度；取1μL文库进行安捷伦2100的片段大小分析，结果如图4所示，从图中可以看出，PCR扩增出来的DNA目的条带长度与目的片段的大小一致，文库大小也与目的片段大小相符，说明制备的文库合格。

　　本实施例步骤1-5所述实验方案总流程示意图如图2所示。

　　由本实施例获得的新冠病毒全长扩增文库在PacBio Sequel II测序平台上进行混合测序，每个样本获得了3G的数据，说明文库质量合格且可获得符合测序要求的数据。

　　实施例2：通过扩增后的测序数据中的病毒数据比例对比

　　由口腔拭子提取的核酸样本WD1、WD2通过实施例1的扩增前后，测序数据中的病毒数据比例如表6所示。

　　表6：

　　由此可见，通过提高本发明提供的扩增方法对样本进行扩增后，数据病毒比例提升至几百倍至几十万倍，可以对病毒信息进行富集，极大的降低了测序数据的浪费。

　　本发明并不仅仅限于说明书和实施方式中所描述，因此对于熟悉领域的人员而言可容易地实现另外的优点和改进，故在不背离权利要求及等同范围所限定的一般概念的精神和范围的情况下，本发明并不限于特定的细节、代表性的方案和这里示出与描述的图示示例。

　　序列表

　　<110> 武汉菲沙基因信息有限公司

　　<120> 一种基于新冠病毒的全基因组全长扩增的三代建库测序方法

　　<141> 2020-06-04

　　<160> 61

　　<170> SIPOSequenceListing 1.0

　　<210> 1

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　　<212> RNA

　　<213> 新冠病毒一致性序列(2019-nCoV)

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　　gcacaagcug augaguacga acuuauguac ucauucguuu cggaagagac agguacguua 26280

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　　guuuggugga cccucagauu caacuggcag uaaccagaau ggagaacgca guggggcgcg 28380

　　aucaaaacaa cgucggcccc aagguuuacc caauaauacu gcgucuuggu ucaccgcucu 28440

　　cacucaacau ggcaaggaag accuuaaauu cccucgagga caaggcguuc caauuaacac 28500

　　caauagcagu ccagaugacc aaauuggcua cuaccgaaga gcuaccagac gaauucgugg 28560

　　uggugacggu aaaaugaaag aucucagucc aagaugguau uucuacuacc uaggaacugg 28620

　　gccagaagcu ggacuucccu auggugcuaa caaagacggc aucauauggg uugcaacuga 28680

　　gggagccuug aauacaccaa aagaucacau uggcacccgc aauccugcua acaaugcugc 28740

　　aaucgugcua caacuuccuc aaggaacaac auugccaaaa ggcuucuacg cagaagggag 28800

　　cagaggcggc agucaagccu cuucucguuc cucaucacgu agucgcaaca guucaagaaa 28860

　　uucaacucca ggcagcagua ggggaacuuc uccugcuaga auggcuggca auggcgguga 28920

　　ugcugcucuu gcuuugcugc ugcuugacag auugaaccag cuugagagca aaaugucugg 28980

　　uaaaggccaa caacaacaag gccaaacugu cacuaagaaa ucugcugcug aggcuucuaa 29040

　　gaagccucgg caaaaacgua cugccacuaa agcauacaau guaacacaag cuuucggcag 29100

　　acguggucca gaacaaaccc aaggaaauuu uggggaccag gaacuaauca gacaaggaac 29160

　　ugauuacaaa cauuggccgc aaauugcaca auuugccccc agcgcuucag cguucuucgg 29220

　　aaugucgcgc auuggcaugg aagucacacc uucgggaacg ugguugaccu acacaggugc 29280

　　caucaaauug gaugacaaag auccaaauuu caaagaucaa gucauuuugc ugaauaagca 29340

　　uauugacgca uacaaaacau ucccaccaac agagccuaaa aaggacaaaa agaagaaggc 29400

　　ugaugaaacu caagccuuac cgcagagaca gaagaaacag caaacuguga cucuucuucc 29460

　　ugcugcagau uuggaugauu ucuccaaaca auugcaacaa uccaugagca gugcugacuc 29520

　　aacucaggcc uaaacucaug cagaccacac aaggcagaug ggcuauauaa acguuuucgc 29580

　　uuuuccguuu acgauauaua gucuacucuu gugcagaaug aauucucgua acuacauagc 29640

　　acaaguagau guaguuaacu uuaaucucac auagcaaucu uuaaucagug uguaacauua 29700

　　gggaggacuu gaaagagcca ccacauuuuc accgaggcca cgcggaguac gaucgagugu 29760

　　acagugaaca augcuaggga gagcugccua uauggaagag cccuaaugug uaaaauuaau 29820

　　uuuaguagug cuauccccau gugauuuuaa uagcuucuua ggagaaugac aaaaaaaaaa 29880

　　aaaaaaaaaa aaaaaaaaa 29899

　　<210> 2

　　<211> 21

　　<212> DNA