| WANG Yong,GAO Zhaoming,XU Ying,LI Guangyu,HE Lisheng,QIAN Peiyuan. 2016. An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community. Acta Oceanologica Sinica, 35(2):131－136 |
| An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community |
| MALBAC技术扩增微生物群落基因组的效率评估 |
| Received:August 20, 2014 Revised:December 26, 2014 |
| DOI：10.1007/s13131-015-0781-x |
| Key words:bacterial DNA MALBAC metagenome amplification |
| 中文关键词: 细菌基因组 MALBAC 宏基因组扩增 |
| 基金项目: |
| Author Name | Affiliation | E-mail | | WANG Yong | Institute of Deep Sea Science and Engineering, Chinese Academy of Sciences (CAS), Sanya 572000, China
Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China | | | GAO Zhaoming | Institute of Deep Sea Science and Engineering, Chinese Academy of Sciences (CAS), Sanya 572000, China
Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China | | | XU Ying | Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China
School of Life Science, Shenzhen University, Shenzhen 518000, China | | | LI Guangyu | Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, China | | | HE Lisheng | Institute of Deep Sea Science and Engineering, Chinese Academy of Sciences (CAS), Sanya 572000, China
Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China | | | QIAN Peiyuan | Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China | boqianpy@ust.hk |
|
| Hits: 2281 |
| Download times: 1563 |
| Abstract: |
| The low biomass in environmental samples is a major challenge for microbial metagenomic studies. The amplification of a genomic DNA was frequently applied to meeting the minimum requirement of the DNA for a high-throughput next-generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles (MALBAC) kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms is examined. The DNA template of 10 pg in each reaction of the MALBAC amplification may generate enough DNA for Illumina sequencing. Using 10 pg and 100 pg templates for each reaction set, the MALBAC kit shows a stable and homogeneous amplification as indicated by the highly consistent coverage of the reads from the two amplified samples on the contigs assembled by the original unamplified sample. Although GenomePlex whole genome amplification kit allows one to generate enough DNA using 100 pg of template in each reaction, the minority of the mixed bacterial species is not linearly amplified. For both of the kits, the GC-rich regions of the genomic DNA are not efficiently amplified as suggested by the low coverage of the contigs with the high GC content. The high efficiency of the MALBAC kit is supported for the amplification of environmental microbial DNA samples, and the concerns on its application are also raised to bacterial species with the high GC content. |
| 中文摘要: |
| 环境样品的低生物量是微生物宏基因组学研究面临的首要挑战,通过基因组扩增技术来满足高通量测序对DNA样品量的需求是最常用的解决策略。MALBAC(Multiple Annealing and Looping Based Amplification Cycles)基因组扩增试剂盒最初为扩增和研究哺乳动物的单细胞基因组而研发。本文中,我们通过人工构建的微生物群落来检测该试剂盒在微生物宏基因组扩增方面的效率和应用可行性。结果表明,每个标准反应中,10 pg的DNA模板量足以满足MALBAC试剂盒对样品扩增的需要。每个标准反应DNA模板用量为10和100 pg时,所扩增DNA样品的基因组覆盖度与原始未扩增样品表现出高度的一致性,证明MALBAC试剂盒扩增效果的高度稳定性和一致性。常用的GenomePlex全基因组扩增试剂盒使我们可以在每个标准反应DNA模板量为100 pg的条件下扩增获得足够的DNA样品,但是结果表明该参照试剂盒无法有效的实现对群落中低丰度细菌菌株基因组的线性扩增。对于MALBAC试剂盒和参照试剂盒而言,在扩增高GC含量的微生物物种基因组DNA方面效率低下。我们的实验结果表明MALBAC试剂盒在高效扩增环境样品宏基因组DNA方面的可行性,但对该试剂盒在扩增环境样品中高GC含量微生物物种方面的适用性存在疑虑。 |
|
HTML
View Full Text
View/Add Comment Download reader |
| Close |
|
|
|
