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青岛能源所开发出高覆盖度的单细胞拉曼分选-测序技术

阅读量：17

发布时间：2021-08-25

来源：酯香香化学采购网

　　【化工仪器网科技前沿】拉曼激活细胞分选耦合测序是菌群单细胞分析的重要手段，但其低基因组覆盖度一直困扰着业界。中国科学院青岛生物能源与过程研究所单细胞中心发明了全新的单细胞拉曼分选-测序技术RAGE-Seq，实现了菌群中单个目标细胞近100%覆盖度的精准测序。该工作发表于Small。

　　微生物组是地球上能量与元素循环的主要载体，与人体、动植物以及环境的健康也息息相关。因此，微生物组的探测、理解和利用，将跨越“尚难培养微生物屏障”，带来生命科学及其应用的共性突破。单细胞拉曼光谱能够无需培养而快速识别菌群中细胞的各种代谢功能(Biotechnol Adv, 2019)，但是一直以来，针对菌群的单细胞拉曼分选和测序都面临着两个瓶颈：首先，如何无损快速地获取特定拉曼表型的单个细胞；其次，如何高覆盖度地获得精确到一个细胞的基因组。

　　针对上述问题，青岛能源所单细胞中心徐腾、公衍海、苏晓璐等发明了一种在液相环境中测量与分选菌群中目标微生物单细胞的拉曼分选-测序耦合技术RAGE-Seq(Raman-activated Gravity-driven Encapsulation and Sequencing；图1)。它通过联用光镊及微流控液滴技术，将特定拉曼表型的细菌单细胞从群体中精准分离，并包裹到皮升级液滴中，然后采用宏观移液的方式便可将包裹有目标细胞的液滴导出转移到试管中，从而快速、精确、简便地实现“单个细胞-单个液滴-单个试管”的拉曼分选流程，可直接耦合下游细胞培养或基因组分析。

　　由于目标细胞在分选过程中始终包裹于水相环境中，RAGE-Seq有效降低了拉曼分选中激光引入的细胞损伤。其导出的单细胞液滴体系，经过简单震荡后可转变成乳化扩增体系，大幅度降低了传统单细胞基因扩增(如单细胞MDA)中存在的偏好性，从而可从一个细胞得到近乎完整的全基因组信息。此外，其独特的单液滴分选转移形式，有效解决了单细胞分析中常见的污染问题。

　　基于单细胞中心前期发明的重水孵育单细胞拉曼药敏快检技术(Anal Chem, 2017)，研究人员用RAGE-Seq从临床尿液样本中直接识别和分选出耐受特定抗生素的临床大肠杆菌，并进行了精确到一个细胞的全基因组测序，覆盖度可达99.5%。这一高覆盖度保证了基因组上所有耐药基因突变均得以全面、精确地揭示。

　　从菌群中直接、精准地获取一个细菌细胞的药敏性表型及其完整基因组，以往还未有先例。因此，RAGE-Seq的这一独特能力，预计将带来临床感染诊断和用药、耐药性传播监控与机制、海洋生态监控与资源挖掘等领域的一系列突破。

　　基于RAGE-Seq，单细胞中心推出了临床单细胞拉曼药敏快检仪CAST-R，以及和现有荧光/明场显微镜耦合的模块式单细胞微液滴分离系统EasySort。这些原创的系列仪器正在服务各种场景下的单细胞功能分选与测序(图1)。

　　该工作由单细胞中心马波和徐健主持完成。该工作得到中科院战略性先导专项、国家自然科学基金委、山东省自然科学基金委的资助。

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[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 2 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000159s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 7 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000175s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 16 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000154s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 17 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000178s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` IN (34,92) AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000164s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` IN (95,38) AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000175s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 1 AND `status` = 1 [ RunTime:0.000141s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 1 [ RunTime:0.000165s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,1,%' ) [ RunTime:0.000163s ]
[ SQL ] SHOW COLUMNS FROM `yzn_goods` [ RunTime:0.000606s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (25,26,29,30,48,1) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000932s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 2 AND `status` = 1 [ RunTime:0.000216s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 2 [ RunTime:0.000163s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,2,%' ) [ RunTime:0.000160s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (42,43,2) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000858s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 7 AND `status` = 1 [ RunTime:0.000173s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 7 [ RunTime:0.000176s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,7,%' ) [ RunTime:0.000151s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (10,14,32,33,49,50,51,52,53,54,55,56,57,58,96,97,7) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000882s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 16 AND `status` = 1 [ RunTime:0.000175s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 16 [ RunTime:0.000176s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,16,%' ) [ RunTime:0.000157s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (59,60,61,62,63,16) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000855s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 17 AND `status` = 1 [ RunTime:0.000185s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 17 [ RunTime:0.000156s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,17,%' ) [ RunTime:0.000192s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (40,64,65,66,67,68,100,17) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000859s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 34 AND `status` = 1 [ RunTime:0.000178s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 92 AND `status` = 1 [ RunTime:0.000154s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` IN (34,92) [ RunTime:0.000183s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,34,%' or arrparentid like '0,92,%' ) [ RunTime:0.000185s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (35,70,71,93,94,34,92) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000876s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 95 AND `status` = 1 [ RunTime:0.000182s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 38 AND `status` = 1 [ RunTime:0.000150s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` IN (95,38) [ RunTime:0.000210s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,38,%' or arrparentid like '0,95,%' ) [ RunTime:0.000172s ]
[ SQL ] SELECT `g`.`name`,p.id as p_id,`p`.`price`,`p`.`type`,`p`.`thumb`,`p`.`unit`,`p`.`is_where`,`p`.`goods_id`,`p`.`repeat_type` FROM `yzn_goods` `g` LEFT JOIN `yzn_goods_property` `p` ON `p`.`goods_id`=`g`.`id` WHERE `p`.`status` = 1 AND `p`.`is_del` = 0 AND `g`.`is_del` = 0 AND `p`.`is_where` LIKE '%1%' AND `g`.`cate_id` IN (47,74,75,76,77,78,79,80,81,82,83,99,95,38) AND `p`.`hot_sale` = '1' GROUP BY `p`.`repeat_type`,`p`.`goods_id` ORDER BY `p`.`sort` DESC,`p`.`id` DESC LIMIT 0,10 [ RunTime:0.000879s ]
[ SQL ] SHOW COLUMNS FROM `yzn_article` [ RunTime:0.000751s ]
[ SQL ] SELECT `A`.*,`AD`.*,`C`.`catname` FROM `yzn_article` `A` INNER JOIN `yzn_article_data` `AD` ON `A`.`id`=`AD`.`did` INNER JOIN `yzn_category` `C` ON `C`.`id`=`A`.`catid` WHERE `A`.`id` = 5705 AND `A`.`status` = 1 LIMIT 1 [ RunTime:0.000613s ]
[ SQL ] SHOW COLUMNS FROM `yzn_category` [ RunTime:0.000553s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` = 35 AND `status` = 1 [ RunTime:0.000169s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` IN (36,37,38,39,40,41,42) AND `status` = 1 [ RunTime:0.000162s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` = 43 AND `status` = 1 [ RunTime:0.000134s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` IN (44,45,46,47,48,49,50) AND `status` = 1 [ RunTime:0.000139s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` = 6 AND `status` = 1 [ RunTime:0.000124s ]
[ DB ] CONNECT:[ UseTime:0.000231s ] mysql:host=127.0.0.1;port=3306;dbname=yzncms;charset=utf8
[ SQL ] SELECT `A`.`id`,`A`.`title` FROM `yzn_article` `A` INNER JOIN `yzn_category` `C` ON `C`.`id`=`A`.`catid` WHERE `A`.`status` = 1 AND `A`.`catid` IN (35,36,37,38,39,40,41,42) ORDER BY `A`.`flag` DESC,`A`.`listorder` ASC,`A`.`updatetime` DESC [ RunTime:0.020379s ]
[ SQL ] UPDATE `yzn_article` SET `hits` = `hits` + 1 WHERE `id` = 5705 [ RunTime:0.000311s ]
[ SQL ] SELECT * FROM `yzn_poster` WHERE `cate_id` = 10 AND `p_c_id` = 41 ORDER BY `listorder` ASC LIMIT 4 [ RunTime:0.000363s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `yzn_article` `a` INNER JOIN `yzn_category` `c` ON `a`.`catid`=`c`.`id` WHERE `catid` = 38 AND `a`.`status` = 1 [ RunTime:0.002606s ]
[ SQL ] SELECT `a`.*,`c`.`catname` FROM `yzn_article` `a` INNER JOIN `yzn_category` `c` ON `a`.`catid`=`c`.`id` WHERE `catid` = 38 AND `a`.`status` = 1 ORDER BY `flag` DESC,`listorder` ASC,`updatetime` DESC LIMIT 0,6 [ RunTime:0.004669s ]
[ SQL ] SELECT COUNT(*) AS tp_count FROM `yzn_article` `a` INNER JOIN `yzn_category` `c` ON `a`.`catid`=`c`.`id` WHERE `catid` = 37 AND `a`.`status` = 1 [ RunTime:0.002591s ]
[ SQL ] SELECT `a`.*,`c`.`catname` FROM `yzn_article` `a` INNER JOIN `yzn_category` `c` ON `a`.`catid`=`c`.`id` WHERE `catid` = 37 AND `a`.`status` = 1 ORDER BY `flag` DESC,`listorder` ASC,`updatetime` DESC LIMIT 0,6 [ RunTime:0.004912s ]