Technical considerations for reduced representation bisulfite sequencing with multiplexed libraries.
Technical considerations for reduced representation bisulfite sequencing with multiplexed libraries.
复制标题
DOI：
10.1155/2012/741542
复制
发表时间：
2012
影响因子：
--
通讯作者：
Morison IM
中科院分区：
文献类型：
--
作者：
Chatterjee A;Rodger EJ;Stockwell PA;Weeks RJ;Morison IM
简化代表性亚硫酸氢盐测序（RRBS）将亚硫酸氢盐转化和下一代测序相结合，是一种创新方法，其特异性富集具有高密度潜在甲基化位点的基因组区域，并能够在单核苷酸分辨率下研究DNA甲基化。Illumina DNA样品制备方案和测序技术的最新进展极大地提高了测序通量能力。尽管新的Illumina技术现在被广泛使用，但与该平台上的多路复用RRBS文库相关的独特挑战先前尚未描述。我们已经对RRBS文库制备方案进行了修改，以对Illumina HiSeq 2000的单个流动池泳道上的多重文库进行测序。此外，我们的分析结合了专门设计用于处理亚硫酸氢盐转化的测序读数并评估从多重文库生成的测序数据的输出和质量的生物信息学管道。对于每个流动池泳道，我们获得了每个样品平均4200万个双端读数，具有与参考人类基因组的高独特映射效率。在这里，我们提供了一个路线图的修改，策略，和故障排除方法，我们实现了优化测序的多重库的RRBS背景。
Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background.
登录
查看更多内容
DOI：
10.1016/j.bbrc.2008.09.044
发表时间：
2008-11-28
影响因子：
3.1
作者：
Igarashi, Jun;Muroi, Satomi;Kawashima, Hiroyuki;Wang, Xiaofei;Shinojima, Yui;Kitamura, Eiko;Oinuma, Toshinori;Nemoto, Norimichi;Song, Fei;Ghosh, Srimoyee;Held, William A.;Nagase, Hiroki
通讯作者：
Nagase, Hiroki
影响因子：
30.8
作者：
通讯作者：
--
影响因子：
64.8
作者：
WRIGHT, CS;ALDEN, RA;KRAUT, J
通讯作者：
KRAUT, J
影响因子：
4.5
作者：
Gertz J;Varley KE;Reddy TE;Bowling KM;Pauli F;Parker SL;Kucera KS;Willard HF;Myers RM
通讯作者：
Myers RM
影响因子：
4.8
作者：
Smith, Zachary D.;Gu, Hongcang;Bock, Christoph;Gnirke, Andreas;Meissner, Alexander
通讯作者：
Meissner, Alexander