Department of Otolaryngology-Head & Neck Surgery, University of Iowa Carver College of Medicine, Iowa City, IA, 52242, USA.
BMC Biotechnol. 2012 May 4;12:20. doi: 10.1186/1472-6750-12-20.
Solution-based targeted genomic enrichment (TGE) protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1) modifying or eliminating time consuming steps; 2) increasing yield to reduce input DNA and excessive PCR cycling; and 3) enhancing reproducible.
We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company.
This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.
基于溶液的靶向基因组富集（TGE）方案允许大规模平行地对感兴趣的基因组区域进行选择性测序。这些方案可以通过以下方式改进：1）修改或消除耗时的步骤；2）提高产量以减少输入 DNA 和过多的 PCR 循环；3）提高可重复性。
我们开发了一种基于溶液的 TGE 方法，用于非自动化工作流程中的下游 Illumina 测序，在杂交后扩增过程中添加标准的 Illumina 条码索引，以允许在测序前进行样品混合。该方法利用 Agilent SureSelect 诱饵、引物和杂交试剂进行捕获，使用现成的试剂进行文库制备步骤，并直接从寡核苷酸制造公司购买用于 Illumina 配对末端测序的接头寡核苷酸。
这种用于 Illumina 测序的基于溶液的 TGE 方法针对小型或中型实验室进行了优化，并通过减少所需的输入 DNA 量、提高捕获产量、优化效率和提高可重复性来解决标准方案的弱点。
