单管共标签长片段读取技术及其在基因组研究中的应用

doi:10.3877/cma.j.issn.2095-1221.2022.02.010

单管长片段读取技术（stLFR）是指在单管中将来源于同一脱氧核糖核酸（DNA）长分子的短读测序片段标记上相同分子标签的无分隔长片段读取技术。通过该技术制备的文库最终产生短读长（200 ~ 1 000 bp）片段进行测序，经过共标签分析后又能拼接成长片段序列，理论上有类似于三代测序进行长片段分析的功能。目前该技术主要用来对目标样本进行基因组装，相关报道在人类、植物、动物和微生物等都有所涉及，但该技术还未得到充分的利用。本文将对基于共标签的stLFR技术及其相关应用进行综述，为解决基因组长片段功能研究提供参考。

关键词: 单管长片段读取, 基因组装, 染色体结构变异, 单细胞转录组测序

Single tube long fragment read (stLFR) refers to an unseparated long fragment reading technique, which can attach to the read sequencing fragments from the same long DNA molecule with the same molecular label. The library can be prepared by this technology and it may generate short read length (200 ~ 1 000 bp) fragments for sequencing. After co-barcoding analysis, it can splice long fragment sequences. Therefore, it is capable of long fragment analysis similarly to the third-generation sequencing in theory. At present, the technology is mainly used to assemble target samples, and studies have been reported in humans, plants, animals, microorganisms, and so on. However, the technology has not been fully utilized. Therefore, the review of co-barcoding stLFR can provide references for solving the functional study of long genome fragments.

Key words: Single tube long fragment read, Gene assembly, Chromosomal structural variation, scRNA-seq

图1 LFR技术建库及组装原理^[10]注：HMM genomic DNA为高分子量基因组脱氧核苷酸；Parent 1/Parent 2为亲本一/亲本二；Wells为样本孔；Barcode adapters为标签序列接头

图2 stLFR文库构建流程示意图^[13]注：Transposome为转座复合体；Transposase为转座酶；Transposon为转座子；Long genomic DNA为长分子基因组脱氧核糖核酸；Clonal barcoded beads为克隆的标签珠子；Transposon inserted DNA为插入转座子的脱氧核糖核酸；Hybridize DNA with beads为脱氧核糖核酸与珠子杂交；PCR primer annealing site为聚合酶链式反应引物退火位点；Capture splint oligo为低聚核苷酸捕获夹板；CapturedgDNA为被捕获的基因组脱氧核糖核酸；Ligate with barcoded oligo为与低聚核苷酸连接；Remove transposase为移除转座酶；Digest excessive oligos为消化过量的低聚核苷酸；ligate 2ndadaptor为连接第二接头；PCR amplification为聚合酶链式扩增反应；Circularization为环化

表1 基于单管共标签技术不同物种基因组装重要参数的统计

物种名称	Contig N50	Scaffold N50	基因功能注释	基因组完整性评估	测序深度
有袋类负鼠	56.91 kb	683.53 Mb	83.90﹪	88.10﹪	100x
圆斑星鲽	79.80 kb	23.80 Mb	95.00﹪	97.20﹪	214.72x
GM12878细胞系	99.78 kb	29.02 Mb	—	91.00﹪	44x
澳洲坚果^[19]	35.60 kb	3.54 Mb	—	88.30﹪	96x
草地贪叶蛾	91.97 kb	0.51 Mb	85.48﹪	95.00﹪	205x
驼背豚	1.78 Mb	15.80 Mb	90.70﹪	95.70﹪	—
冰川红杆菌^[22]	2.80 Mb	4.60 Mb	99.97﹪	99.97﹪	500x
刺鲀^[23]	70.84 kb	6.10 Mb	87.72﹪	95.70﹪	219.8x
湄公河虎鲈^[24]	171.23 kb	9.73 Mb	—	95.19﹪	121x

表1 基于单管共标签技术不同物种基因组装重要参数的统计

物种名称	Contig N50	Scaffold N50	基因功能注释	基因组完整性评估	测序深度
有袋类负鼠	56.91 kb	683.53 Mb	83.90﹪	88.10﹪	100x
圆斑星鲽	79.80 kb	23.80 Mb	95.00﹪	97.20﹪	214.72x
GM12878细胞系	99.78 kb	29.02 Mb	—	91.00﹪	44x
澳洲坚果^[19]	35.60 kb	3.54 Mb	—	88.30﹪	96x
草地贪叶蛾	91.97 kb	0.51 Mb	85.48﹪	95.00﹪	205x
驼背豚	1.78 Mb	15.80 Mb	90.70﹪	95.70﹪	—
冰川红杆菌^[22]	2.80 Mb	4.60 Mb	99.97﹪	99.97﹪	500x
刺鲀^[23]	70.84 kb	6.10 Mb	87.72﹪	95.70﹪	219.8x
湄公河虎鲈^[24]	171.23 kb	9.73 Mb	—	95.19﹪	121x

图3 stLFR数据分析复杂结构易位结果分析图^[13]注：a图为染色体平衡易位分析图谱，b图为染色体倒位分析图谱

表2 单管共标签长片段读取技术与二、三代测序平台的多方面比较

	NGS	ONT-nanopore	PacBio-SMRT	MGI-stLFR	Nanopore+ SMRT	Nanopore+ tLFR	SMRT+ tLFR
覆盖率评估	最低	中等	较高	较高	> 90﹪	> 90﹪	> 90﹪
错误率评估	最低	最高	略高于NGS	略高于NGS	中等	略高于NGS	略高于NGS
样本获得成本	最低	略高于NGS	最高	较高	—	—	—
建库成本评估	最低	较高	较高	略高于NGS	—	—	—
测序成本评估	最低	最高	最高	与NGS相当	—	—	—

表2 单管共标签长片段读取技术与二、三代测序平台的多方面比较

	NGS	ONT-nanopore	PacBio-SMRT	MGI-stLFR	Nanopore+ SMRT	Nanopore+ tLFR	SMRT+ tLFR
覆盖率评估	最低	中等	较高	较高	> 90﹪	> 90﹪	> 90﹪
错误率评估	最低	最高	略高于NGS	略高于NGS	中等	略高于NGS	略高于NGS
样本获得成本	最低	略高于NGS	最高	较高	—	—	—
建库成本评估	最低	较高	较高	略高于NGS	—	—	—
测序成本评估	最低	最高	最高	与NGS相当	—	—	—

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Application of co-barcoding single tube long fragment read technology in genome research

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