Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.
Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.
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DOI：
10.1038/s41598-017-05436-4
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发表时间：
2017-07-12
影响因子：
4.6
通讯作者：
Takeyama H
中科院分区：
文献类型：
--
作者：
Hosokawa M;Nishikawa Y;Kogawa M;Takeyama H
需要大规模并行单细胞基因组测序来进一步了解复杂生物系统中的遗传多样性。全基因组扩增（WGA）是单细胞测序的第一步，但其通量和准确性在常规反应平台中不足。在这里，我们介绍单液滴多重位移扩增（sd-MDA），这种方法能够大规模并行扩增单细胞基因组，同时保持序列准确性和特异性。数以万计的单细胞被分成数百万皮升液滴，然后通过微流体通道中的被动液滴融合进行裂解和 WGA。 Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination.这使得能够从单细胞（21,000 个单细胞/小时）高通量采集无污染和细胞特异性序列读数，从而与传统方法相比提高了序列数据质量。这种方法可以对单个细菌细胞和人类癌细胞进行全基因组分析。所获得的测序覆盖度可与传统技术相媲美，并具有卓越的序列质量。此外，我们还演示了未培养的土壤细菌的从头组装，并通过单细胞测序获得了基因组草案。这种 sd-MDA 有望在单细胞测序中灵活且可扩展地使用。
Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.
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影响因子：
4.5
作者：
Marcy, Yann;Ishoey, Thomas;Lasken, Roger S.;Stockwell, Timothy B.;Walenz, Brian P.;Halpern, Aaron L.;Beeson, Karen Y.;Goldberg, Susanne M. D.;Quake, Stephen R.
通讯作者：
Quake, Stephen R.
影响因子：
3.7
作者：
Dichosa, Armand E. K.;Fitzsimons, Michael S.;Han, Cliff S.
通讯作者：
Han, Cliff S.
DOI：
10.1038/nrg3785
发表时间：
2014-09
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
通讯作者：
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SNES：单核外显子组测序。
影响因子：
12.3
作者：
Leung ML;Wang Y;Waters J;Navin NE
通讯作者：
Navin NE
影响因子：
12.3
作者：
Navin NE
通讯作者：
Navin NE