Ultra-precise detection of mutations by droplet-based amplification of circularized dna
文献类型：期刊论文
| 作者 | Wang,Kaile1,2; Ma,Qin1,2; Jiang,Lan1; Lai,Shujuan1; Lu,Xuemei1; Hou,Yali1; Wu,Chung-I1,3,4; Ruan,Jue1,5 |
| 刊名 | Bmc genomics
 |
| 出版日期 | 2016-03-09 |
| 卷号 | 17期号:1 |
| 关键词 | Accurate sequencing Low frequency mutation detection Low bias amplification Droplet (water in oil) based amplification Low input ngs library |
| ISSN号 | 1471-2164 |
| DOI | 10.1186/s12864-016-2480-1 |
| 通讯作者 | Hou,yali(houyl@big.ac.cn) ; Wu,chung-i(cw16@uchicago.edu) ; Ruan,jue(ruanjue@gmail.com) |
| 英文摘要 | Abstractbackgroundngs (next generation sequencing) has been widely used in studies of biological processes, ranging from microbial evolution to cancer genomics. however, the error rate of ngs (0.1?%?~?1?%) is still remaining a great challenge for comprehensively investigating the low frequency variations, and the current solution methods have suffered severe amplification bias or low efficiency.resultswe creatively developed droplet-cirseq for relatively efficient, low-bias and ultra-sensitive identification of variations by combining millions of picoliter uniform-sized droplets with cir-seq. droplet-cirseq is entitled with an incredibly low error rate of 3?~?5 x 10-6. to systematically evaluate the performances of amplification uniformity and capability of mutation identification for droplet-cirseq, we took the mixtures of two e. coli strains as specific instances to simulate the circumstances of mutations with different frequencies. compared with cir-seq, the coefficient of variance of read depth for droplet-cirseq was 10 times less (p?=?2.6 x 10-3), and the identified allele frequency presented more concentrated to the authentic frequency of mixtures (p?=?4.8 x 10-3), illustrating a significant improvement of amplification bias and accuracy in allele frequency determination. additionally, droplet-cirseq detected 2.5 times genuine snps (p??a, c-?>?t). our findings indicated that 30?pg dna input accommodated in 5?~?10 million droplets resulted in maximal detection of authentic mutations compared to 3?pg (p?=?1.2 x 10-8) and 300?pg input (p?=?2.2 x 10-3).conclusionswe developed a method namely droplet-cirseq to significantly improve the amplification bias, which presents obvious superiority over the currently prevalent methods in exploitation of ultra-low frequency mutations. droplet-cirseq would be promisingly used in the identification of low frequency mutations initiated from extremely low input dna, such as dna of uncultured microorganisms, captured dna of target region, circulation dna of plasma et al, and its creative conception of rolling circle amplification in droplets would also be used in other low input dna amplification fields. |
| 语种 | 英语 |
| WOS记录号 | BMC:10.1186/S12864-016-2480-1 |
| 出版者 | BioMed Central |
| URI标识 | http://www.irgrid.ac.cn/handle/1471x/2374305 |
| 专题 | 中国科学院大学 |
| 通讯作者 | Hou,Yali; Wu,Chung-I; Ruan,Jue |
| 作者单位 | 1.Chinese Academy of Sciences; Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics 2.University of Chinese Academy of Sciences 3.Sun Yat-Sen University; State Key Laboratory of Biocontrol, School of Life Sciences 4.University of Chicago; Department of Ecology and Evolution 5.Chinese Academy of Agricultural Sciences; Agricultural Genomics Institute at Shenzhen |
| 推荐引用方式 GB/T 7714 | Wang,Kaile,Ma,Qin,Jiang,Lan,et al. Ultra-precise detection of mutations by droplet-based amplification of circularized dna[J]. Bmc genomics,2016,17(1). |
| APA | Wang,Kaile.,Ma,Qin.,Jiang,Lan.,Lai,Shujuan.,Lu,Xuemei.,...&Ruan,Jue.(2016).Ultra-precise detection of mutations by droplet-based amplification of circularized dna.Bmc genomics,17(1). |
| MLA | Wang,Kaile,et al."Ultra-precise detection of mutations by droplet-based amplification of circularized dna".Bmc genomics 17.1(2016). |
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来源：中国科学院大学
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