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在NGS建库过程中,如何避免常见的操作失误
发布时间:2019-08-26   点击次数:150次

2013年,哈佛医学院的研究人员曾在《Nature Review Genetics》杂志上发表了一篇很有意思的综述,讨论了新一代测序(NGS)实验中错误的来源1彩搜网。文章指出,在样品制备和文库制备环节,用户操作失误(user error)都是常有的事,比如贴错标签或者加错溶液。

彩搜网RANER,CEXUBIJINGBUTONGYUHESUANCHUNHUA,HOUZHESHIBAILEDABULEZHONGZUO。DUICEXUERYAN,YIGEJIANDANDEYIYEHUOHUNHECUOWUYOUKENENGRANGNIFUCHUGAOANGDEDAIJIA。WULUNLAOBANSHIFOUnice,CEXUSHIYANCONGTOUZAILAIDEGANSHOUKENDINGBUJIA,SHIYANSHIXUYAOHAOFEIDALIANGDESHIJIANHECHENGBEN,KUANGQIEYOUXIEYANGBENHAIHENXIYOU。BUGUO,MEICIYAOCHULINEIMEDUODEYANGBEN,JISHIDAQISHIERFENJINGSHEN,HAISHINANBAOBUCHUCUO。

WEILEBIMIANCAOZUOSHIWUDEFASHENG,SAIMOFEIXIANGDAOLEYIGEJUEMIAODEHAOBANFA。Invitrogen CollibriXILIEWENKUZHIBEISHIJIHEZHONGDEMEIZHONGSHIJIDOUDAIYOUYIZHONGSHIZONGRANLIAO,BIANYURENMENQINGXIGUANCHAWENKUZHIBEIGUOCHENG。RUONIKANDAORONGYEJUNYUNBIANSE(BIRUCONGLANSEBIANWEILVSE,TU1),ZEKEYIQUEXINXIANGGUANSHIJIYITIANJIAQIECHEDIHUNYUN。RUOSHILOUJIALEMOUZHONGSHIJI,HUOZHEMEIYOUHUNHEJUNYUN,ZEGUANCHABUDAOYUQIDEYANSEBIANHUA。


彩搜网图1. Collibri PS DNA文库制备试剂盒的颜色变化

BUYONGDANXIN,SHIJIZHONGDEDUOXINGRANLIAOJIBUHUIGANRAOMEICUFANYING,YEBUHUIYINGXIANGCEXUJIEGUO。HOUCHUNHUASUODEDEWENKUSHICHENGQINGTOUMINGDE。ZHEIZHONGSHISHIDEYANSEBIANHUATISHI,RANGCAOZUOSHIWUDEKENENGXINGDADAJIANGDI,YERANGNIDUIWENKUZHIBEIDEGUOCHENGCHONGMANXINXIN。

KANDAOZHEILI,NIYIDINGXIANGZHIDAO,CollibriXILIEWENKUZHIBEISHIJIHEBAOHANNAXIECHANPIN,SHIHENAXIEYINGYONG?QISHI,ZHENDUIQUANJIYINZUCEXUHEZHUANLUZUCEXU,CIXILIEDOUYOUXIANGYINGDESHIJIHETIGONG,JUNSHIYONGYUIlluminaDEGAOTONGLIANGCEXUYI。XIAMIANJIURANGXIAOBIANLAIYIYIJIESHAO。

全基因组测序

▪ Collibri PS DNA文库制备试剂盒适用于物理片段化的基因组,支持1-1,000 ng的样本起始量
▪ Collibri PCR-Free PS DNA文库制备试剂盒同样适用于物理片段化的基因组,采用PCR-free的实验方案,支持500-1,000 ng的样本起始量
▪ Collibri ES DNA文库制备试剂盒适用于酶法片段化的基因组,支持1-500 ng的样本起始量
▪ Collibri PCR-Free ES DNA文库制备试剂盒同样支持酶法片段化的基因组,采用PCR-free的实验方案,支持100-500 ng的样本起始量

GENJUPIANDUANHUAFANGSHIDEBUTONG,Collibri DNAWENKUZHIBEISHIJIHEKEFENWEILIANGDALEI:WULIPIANDUANHUA(PS)HEMEIFAPIANDUANHUA(ES)。WULIFANGFAPIANDUANHUAZHUYAOSHILIYONGJIXIEPOSUIDEFANGFAJIANGJIYINZUSUIJIDADUAN,RUCHAOSHENGPOSUI、WUHUADENG。MEIXUEFANGFAPIANDUANHUAZHUYAOLIYONGMEIDEJIANQIEGONGNENGJIANGJIYINZUPIANDUANHUA。MEIFAPIANDUANHUAFANGSHICAOZUOJIANDAN,DANWULIPIANDUANHUAFANGSHIDEPIANDUANSUIJIXINGGENGHAO。CIWAI,MEIZHONGSHIJIHEDOUYOUPCRKUOZENGCHANPINHEPCR-freeCHANPINKEXUAN。

YUTONGLEICHANPINXIANGBI,CollibriXILIEWENKUZHIBEISHIJIHENENGGOUZAIGENGDUANSHIJIANNEIWANCHENGJIANKU。YICollibri PCR-Free PS DNAWENKUZHIBEISHIJIHEWEILI,WENKUZHIBEIKEZAI1.5XIAOSHINEIWANCHENG,ERZHENGGESHIYANLIUCHENGDEZONGSHIJIANSUODUANZHI6.5XIAOSHI。QITACHANPINYIBANXUYAO7XIAOSHIYISHANG,SHENZHICHAOGUO10XIAOSHI。

RUOQISHICAILIAOYOUXIAN,KEYIXUANZEPCRFANGAN。BUGUANSHIPCR-freeWENKUHAISHIJINGPCRKUOZENGDEWENKU,DOUKEYISHIZHONGBAOCHIJUNYIDEGCFUGAIDU,JIANGGC biasJIANGZHIDI。JISHISHI“CEXUKUNNANHU”,RUbad promotersHUOGCHANLIANGGAODA75%DEQUYU,FUGAIDUYEYOUYUTONGLEICHANPIN,BAOKUOFFPEYANGBEN。ZHEIZHONGGAOFUGAIDUSHIDETUBIANJIANCEDELINGMINDUHEZHUNQUEDUDEDAOZENGQIANG。

彩搜网JINGGUOSHIYANZHENGSHI,Collibri DNAWENKUZHIBEISHIJIHENENGGOUDIJIANCEXINSHENGTUBIAN(De Novo mutations)。BUGUANSHIPCRFANGANHAISHIPCR-freeFANGAN,TAMENDOUNENGGOUJIANCESNP,DUIYU10 ngJIYISHANGDEQISHIDNAYANGBENLIANG,Collibri PS DNAWENKUZHIBEISHIJIHEDESNP recallGAOYU98.8%(TU2)。CIWAI,JISHISHIDIQISHILIANGYANGBEN,CHARUTUBIANJIANCEDEindel recallYEGAOYUTONGLEICHANPIN。

Collibri DNAWENKUZHIBEISHIJIHEBAOHANLEWGSWENKUGOUJIANSUOXUDESUOYOUZUFEN,CHULEHEXINMEIZUFENYIWAI,HAIBAOKUOCIZHUJIJINGTESHUSHEJIDEindexed adaptors,WUXUZAIDANDUGOUMAI。SHIJIHESUIFUTIGONGDUTESHUANGDUANindex(UDI)。YUCHUANTONGDECDIXIANGBI,UDIDESHIYONGDADAJIANGDILEBIAOQIANCHUANRAO(index hopping)DEBILI。


图2. 检测突变

全转录组测序

彩搜网Collibri RNACEXUJIANKULIUCHENGBUTONGYUSHIMIANSHANGYIBANDERNA-SeqJIANKULIUCHENG。RNAJINGMEIPIANDUANHUAHOUZHIJIEYUadaptor(HANYOUFUZHUoligo)LIANJIE,ZHIHOUNIZHUANLUXINGCHENGcDNA,HOUZAITONGGUOPCRKUOZENGYINRUindex(TU3)。

ZHEIZHONGDUTEDEJIANKULIUCHENGKEBAOLIUYANGBENDEFUZADU,SHIYONGYUBUTONGZHILIANGDEYANGBENLEIXING,RUWANZHENGRNA、JIANGJIERNA、FFPEYANGBENDENG。SHIYONGTONGYIYINWUJINXINGcDNAHECHENG,BUHUISHOUGCHANLIANGDEYINGXIANG,QIEWUXUYINRUSUIJIOligoXULIE,BIMIANJIANCESNPHUODIANTUBIANSHICHUXIANJIAYANGXING。


图3. Collibri RNA测序建库流程

彩搜网Collibr™ Stranded RNAWENKUZHIBEISHIJIHETONGYANGNENGGOUSHIXIANKUAISUJIANKU。BAOKUOrRNAQUCHUZAINEI,ZHUANLUZUWENKUGOUJIANKEZAI6.5XIAOSHINEIWANCHENG。ZHEIYANGSHIDECONGRNATIQUDAOWENKUGOUJIANWANCHENGDEZONGSHIJIANSUODUANZHI10.5XIAOSHIZUOYOU。

KEXUANDECollibri H/M/R rRNA Depletion KitHAINENGCONG100-1000 ngREN、XIAOSHUHUODASHUZONGRNAZHONGQUCHUrRNA。YUSHIMIANSHANGQITArRNAQUCHUFANGFABUTONG,ZHEIZHONGSHIJIHEJIYUQINHETANZHENZAJIAOHECIZHUCHUNHUAFANGFA,KETEYIDIQUCHUrRNAXULIE。TANZHENDESHULIANGHEBAXIANGWEIZHIJUNJINGJINGXINSHEJI,KEYIQUCHU28S、18S、5.8S、45S、5S、mt16SHEmt12SHETANGTIXULIE。

彩搜网Collibri RNAWENKUZHIBEISHIJIHENEIBAOHANRNA-SeqWENKUGOUJIANSUOXUDESUOYOUZUFEN,CHULEPIANDUANHUAMEI、RNAMODUANXIUSHISHIJI、Adaptor、LIANJIEMEI、FANZHUANLUSHIJI、WENKUKUOZENGSHIJIWAI,HAIBAOHANrRNAQUCHUSHIJI、CHUNHUACIZHUYIJIindex primerDENG。

文库定量

JIYUqPCRDECollibriWENKUDINGLIANGSHIJIHENENGGOUDUIWENKUYOUXIAONONGDUJINXINGZHUNQUEDINGLIANG,SHIHEDAPILIANGYANGBENDEWENKUDINGLIANG。GAISHIJIHEBAOHANCollibriWENKUDINGLIANGYUHUNYE、Collibri DNABIAOZHUNPINHECollibriWENKUXISHIHUANCHONGYE。ZAIWENKUDINGLIANGSHI,SHIYONGYIZU6GEYUXIANXISHIDEDNACANKAOBIAOZHUNPINLAISHENGCHENGBIAOZHUNQUXIAN,TONGGUOZHEIYIBIAOZHUNQUXIANKECEDINGYANGBENWENKUNONGDU。

JIYUInvitrogen™ Platinum™ II Taq REQIDONGDNAJUHEMEIDEqPCRYUHUNYEHANYOUBAXIANGIllumina P5HEP7 AdaptorXULIEDEYINWU。Platinum II Taq REQIDONGDNAJUHEMEIKEYIJUNYUNKUOZENGGCHANLIANGBUTONGHEZHANGDUANGEYIDEDNAPIANDUAN,YINCISHIDINGLIANGCEXUWENKUDELIXIANGXUANZE。

YUYINGGUANGJIANCEFANGFAXIANGBI,qPCRJIANCEJINCEDINGHANYOUP5HEP7 AdaptorDEWENKUPIANDUAN,YINCINENGGOUGENGJIATEYIHEZHUNQUEDIJIANCENGSCEXUWENKU。CollibriJIANCESHIFENLINGMIN,KEYIDINGLIANGFEIMOERLIANGJIDEPIANDUANWENKU。QISHILIANGYAOQIUJIDI,TONGCHANGJINXU4 μLXISHIDEWENKUYANGBEN,NONGDU>0.0002 pMJIKE。TONGYANG,YANSEZHISHIKEJIANGDIFANYINGPEIZHIQIJIANYIYECUOWUDEFENGXIAN。

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