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青岛能源所开发出二代拉曼激活细胞弹射耦合测序技术

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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】拉曼激活细胞弹射耦合测序(RACE-Seq)是微生物组单细胞分析的手段之一，但其基因组覆盖度通常不超过10%，而且核酸扩增成功率低，极大限制了其应用。中国科学院青岛生物能源与过程研究所单细胞中心系统评估和优化了该技术，并改进了单细胞基因组扩增环节，从而提高了RACE-Seq的基因组覆盖度和核酸扩增成功率。该工作近日发表于Analytical Chemistry。

　　作为微生物在自然界中的存在形式，微生物组(菌群；Microbiome)与人体和自然界的过去、现在和未来息息相关。但是，菌群中绝大部分微生物组分通常难以快速培养和利用，因此，微生物组又被称为"生命暗物质”。如何识别和利用"生命暗物质”中的功能组分，一直是生命科学和环境科学中的一个共性方法学问题。

　　针对这个瓶颈，单细胞中心和业界同行前期发明了一系列单细胞拉曼分选技术与装备(RACS-Seq; Biotechnol Adv, 2019; doi: 10.1016/j.biotechadv.2019.04.010)。RACS-Seq技术家族的工作原理均为，基于单细胞拉曼光谱来从菌群样品中直接识别和分选特定代谢功能的细胞，进而与核酸提取、扩增和测序对接，从而深刻理解菌群中"谁在做什么？如何做？”。其中的RACE-Seq技术将菌群细胞风干后、在弹射芯片表面平铺成单层，逐一采集拉曼光谱后，采用另一束激光将目标拉曼光谱的细胞从芯片表面弹射到接收芯片的小孔中，进而在小孔中进行核酸扩增和测序文库制备。但是，在已发表的RACE-Seq应用于人体或菌群分析的工作中，单细胞基因组测序的覆盖度普遍很低(很少超过20%，大多在10%以下)，同时其完整流程的成功率也不高，以致于通常需要把几个乃至几十个细胞弹射至同一个小孔中，混合进行基因组扩增和测序，以提高实验的成功率。这种低覆盖度而且混合测定的单细胞基因组数据，从根本上阻碍了在细胞"个体”水平的基因组结构分析与代谢功能重建。

　　为了揭示RACE-Seq基因组覆盖率极低的原因，单细胞中心苏晓璐与公衍海带领的研究小组，基于长期努力，从实验操作和数据计算分析两个角度入手，建立了系统性的RACE-Seq方法和芯片的质量评估和控制体系。在此基础上，全面、定量地评估了细胞裂解条件、核酸扩增条件和拉曼测量条件等每一环节的关键参数对RACE-Seq基因组测序质量的影响。结果发现，激光照射强度在拉曼信号采集过程中至关重要，并与单细胞核酸扩增(MDA)的成功率成反比。

　　针对这些问题，研究人员开发出了二代RACE-Seq(图1):在单细胞扩增体系中加入特定油相并充分震荡，产生大量随机包裹的微体积液滴，造成每个DNA片段在破乳前均达到饱和扩增，从而大幅提高RACE-Seq中单细胞基因组的覆盖度。这种操作方法简单快速，而且容易实现自动化。

　　与第一代RACE-Seq相比，该二代技术可将纯培养大肠杆菌(每个MDA体系含5个弹射出的细胞)单细胞基因组覆盖度从 < 20% 提高到50%。针对土壤菌群样品的测试表明(每个MDA体系含2~5个弹射出的细胞)，二代技术可将MDA反应的成功率从58%提高到 90%，而将MDA产物16S扩增成功率从0%大幅提升至83%。基于这些优点，单细胞中心开发了RACE-Seq试剂盒(图2)，以服务于各种环境样品和应用场景。

　　尽管如此，由于拉曼全谱采集对于细胞活性的破坏，RACE-Seq目前还难以实现在"一个”细菌细胞水平的高覆盖度基因组测序或细胞培养。针对这些局限性，研究人员正在开发一系列液相拉曼分选耦合测序器件，以建立与完善一个高拉曼全谱信号质量、高基因组测序覆盖度、高效保护细胞活性、一个细菌细胞精度的微生物组分析技术服务体系。

　　苏晓璐和公衍海是论文的共同第一作者。该工作得到中科院先导B项目、青岛海洋科学与技术试点国家实验室、国家自然科学基金等的支持。

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[ SQL ] SELECT `g`.`name`,`p`.`price`,`p`.`unit`,`p`.`type`,`p`.`thumb`,p.id as p_id,`p`.`value`,`p`.`is_where`,`p`.`sku_unit`,`p`.`repeat_type`,`p`.`goods_id` FROM `yzn_goods_property` `p` LEFT JOIN `yzn_goods` `g` 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 `p`.`recommend` = 1 GROUP BY `p`.`goods_id`,`p`.`repeat_type` ORDER BY `p`.`sort` DESC,`g`.`id` DESC LIMIT 0,15 [ RunTime:0.002459s ]
[ SQL ] SHOW COLUMNS FROM `yzn_goods_category` [ RunTime:0.000859s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `hot_search` = 1 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000366s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `is_show` > 0 AND `status` = 1 ORDER BY `listorder` ASC [ RunTime:0.000258s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `id` IN (1,2,7,16,17,34,92,95,38) [ RunTime:0.000237s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 1 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000182s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 2 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000160s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 7 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000173s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 16 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000164s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 17 AND `status` = 1 ORDER BY `listorder` ASC,`id` DESC [ RunTime:0.000157s ]
[ 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:0.000176s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 1 AND `status` = 1 [ RunTime:0.000150s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 1 [ RunTime:0.000158s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,1,%' ) [ RunTime:0.000161s ]
[ SQL ] SHOW COLUMNS FROM `yzn_goods` [ RunTime:0.000622s ]
[ 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:0.000951s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 2 AND `status` = 1 [ RunTime:0.000198s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 2 [ RunTime:0.000160s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,2,%' ) [ RunTime:0.000176s ]
[ 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:0.000873s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 7 AND `status` = 1 [ RunTime:0.000178s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 7 [ RunTime:0.000160s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,7,%' ) [ RunTime:0.000150s ]
[ 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:0.000917s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 16 AND `status` = 1 [ RunTime:0.000311s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 16 [ RunTime:0.000197s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,16,%' ) [ RunTime:0.000177s ]
[ 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:0.000883s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 17 AND `status` = 1 [ RunTime:0.000181s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` = 17 [ RunTime:0.000159s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,17,%' ) [ RunTime:0.000154s ]
[ 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:0.000860s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 34 AND `status` = 1 [ RunTime:0.000185s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 92 AND `status` = 1 [ RunTime:0.000143s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` IN (34,92) [ RunTime:0.000178s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,34,%' or arrparentid like '0,92,%' ) [ 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 (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:0.000843s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 95 AND `status` = 1 [ RunTime:0.000176s ]
[ SQL ] SELECT `id`,`catname` FROM `yzn_goods_category` WHERE `pid` = 38 AND `status` = 1 [ RunTime:0.000156s ]
[ SQL ] SELECT * FROM `yzn_goods_category` WHERE `status` = 1 AND `id` IN (95,38) [ RunTime:0.000190s ]
[ SQL ] SELECT `id` FROM `yzn_goods_category` WHERE `status` = 1 AND ( arrparentid like '0,38,%' or arrparentid like '0,95,%' ) [ RunTime:0.000179s ]
[ 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:0.000899s ]
[ SQL ] SHOW COLUMNS FROM `yzn_article` [ RunTime:0.000832s ]
[ 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` = 5718 AND `A`.`status` = 1 LIMIT 1 [ RunTime:0.000518s ]
[ SQL ] SHOW COLUMNS FROM `yzn_category` [ RunTime:0.000563s ]
[ 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:0.000149s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` = 43 AND `status` = 1 [ RunTime:0.000141s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` IN (44,45,46,47,48,49,50) AND `status` = 1 [ RunTime:0.000163s ]
[ SQL ] SELECT `id` FROM `yzn_category` WHERE `parentid` = 6 AND `status` = 1 [ RunTime:0.000196s ]
[ DB ] CONNECT:[ UseTime:0.000263s ] 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:0.020366s ]
[ SQL ] UPDATE `yzn_article` SET `hits` = `hits` + 1 WHERE `id` = 5718 [ RunTime:0.000276s ]
[ SQL ] SELECT * FROM `yzn_poster` WHERE `cate_id` = 10 AND `p_c_id` = 41 ORDER BY `listorder` ASC LIMIT 4 [ RunTime:0.000320s ]
[ 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:0.002626s ]
[ 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:0.004572s ]
[ 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:0.002509s ]
[ 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:0.004916s ]