Genome Sequencing by Natural DNA Synthesis on Amplified DNA Clones

通过对扩增的 DNA 克隆进行天然 DNA 合成进行基因组测序

基本信息

批准号：
8119145
负责人：
XIAOHUA HUANG
金额：
$ 61.78万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-19 至 2012-05-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): We propose to combine the best proven aspects of SBS with streamlined methods for DNA amplification and high-speed fluorescence imaging to develop and implement a platform for rapid and inexpensive genome resequencing and de novo sequencing. Our platform is called "Natural Sequencing by Synthesis" (nSBS). Amplified DNA molecular clones will be sequenced in massive parallel by cyclic sequencing by synthesis using DNA polymerases and mostly natural nucleotides. The key is to use a small percentage of a cleavable fluorescently-labeled nucleotide along with the natural nucleotide in the cyclic base-by-base DNA sequencing by synthesis process for sequence detection. Not only will the fluorescently-labeled nucleotide incorporation be sparse but the fluorescent moiety will also be cleaved off after each imaging step. This will minimize the modification of the natural structure of the extending DNA template and ensure that DNA synthesis will not be significantly affected. With this strategy, homopolymer tracts can be sequenced and very long read lengths can be achieved. We present a concept for a new breakthrough technology called natural DNA sequencing by synthesis (nSBS). We also present several other breakthrough innovations: 1) In situ massive parallel amplification of single DNA molecules with micro fabricated arrays and rapid assembly of DNA templates. 2) The usage of an automaton to validate and optimize the new nSBS chemistry for cyclic sequencing by synthesis using DNA polymerases and commercially available nucleotides and nucleotides we will design and synthesize for efficient incorporation; 3) The decoupling of the reaction from detection to make the system scalable to very high-density arrays for whole genome sequencing. Since much higher density arrays can be used and only one enzyme (DNA polymerase) will be used, much less reagent will be needed. This will result in dramatic improvement of throughput and reduction in reagent cost. 4) The implementation of a double barrel paired-end strategy and new algorithms for de novo sequence assembly. In the long run this technology will have a great potential to enable very accurate re-sequencing and de novo sequencing of genomes at high speed and much lower cost for biomedical research and personalized medicine. PROJECT HEALTH RELEVANCE We propose to develop a breakthrough DNA sequencing technology called DNA sequencing by natural DNA synthesis (nSBS). We will combine streamlined methods for genome-scale DNA amplification with the new sequencing chemistry to engineer a sequencing platform for ultra-fast and low-cost human genome sequencing so that routine sequencing of individual human genomes can be performed for biomedical applications and personalized medicine.

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Linear nicking endonuclease-mediated strand-displacement DNA amplification.

DOI：
10.1016/j.ab.2011.02.025
发表时间：
2011-07-01
期刊：
Analytical biochemistry
影响因子：
2.9
作者：
Joneja A;Huang X
通讯作者：
Huang X

Multiplexed protein analysis using encoded antibody-conjugated microbeads.

使用编码的抗体缀合微珠进行多重蛋白质分析。

DOI：
10.1098/rsif.2010.0594
发表时间：
2011
期刊：
Journal of the Royal Society, Interface
影响因子：
0
作者：
Theilacker,Nora;Roller,EricE;Barbee,KristopherD;Franzreb,Matthias;Huang,Xiaohua
通讯作者：
Huang,Xiaohua

Capture and enumeration of mRNA transcripts from single cells using a microfluidic device.