Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Biotechniques. 2012 Jul;53(1):61-2. doi: 10.2144/0000113891.
Advances in sequencing technologies have dramatically reduced costs in producing high-quality draft genomes. However, there are still many contigs and possible misassembled regions in those draft genomes. Improving the quality of these genomes requires an efficient and economical means to close gaps and resequence some regions. Sequencing pooled gap region PCR products with Pacific Biosciences (PacBio) provides a significantly less expensive means for this need. We have developed a genome improvement pipeline with this strategy after decreasing a loading bias against larger PCR products in the PacBio process. Compared with Sanger technology, this approach is not only cost-effective but also can close gaps greater than 2.5 kb in a single round of reactions, and sequence through high GC regions as well as difficult secondary structures such as small hairpin loops.
测序技术的进步极大地降低了生产高质量草图基因组的成本。然而，这些草图基因组中仍然存在许多重叠序列和可能的错误组装区域。要提高这些基因组的质量，需要一种高效、经济的方法来填补缺口并对一些区域进行重测序。使用太平洋生物科学公司（PacBio）对 pooled gap region PCR 产物进行测序，可以满足这一需求，且成本显著降低。在降低 PacBio 过程中对较大 PCR 产物的加载偏倚后，我们采用这种策略开发了一种基因组改进流程。与 Sanger 技术相比，这种方法不仅具有成本效益，而且可以在单个反应轮次中填补大于 2.5kb 的缺口，还可以通过高 GC 区域以及小发夹环等困难的二级结构进行测序。
