A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits
A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits
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DOI：
10.1093/nar/gkv773
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发表时间：
2015-12-15
影响因子：
14.9
通讯作者：
Magiorkinis, Gkikas
中科院分区：
文献类型：
--
作者：
Karamitros, Timokratis;Magiorkinis, Gkikas
在复杂的下一代测序文库中富集靶向区域通常使用生物素化诱饵来捕获所需序列。该方法导致靶及其侧翼区域上的高读段覆盖。Oxford Nanopore Technologies最近发布了一款USB3.0接口的测序仪MinION。到目前为止，还没有标准化用于富集MinION文库的特定方法。在这里，使用生物素化的PCR产生的诱饵在一种新的方法，我们描述了一种简单而有效的方法，用于多重富集的MinION库，克服了与测序接头的化学和DNA片段的长度相关的技术限制。使用噬菌体λ和大肠杆菌作为模型，我们选择性地富集特定靶标，显著增加相应的读取覆盖率，消除不需要的区域。我们表明，通过捕获包含靶序列的基因组片段，我们恢复了延伸靶区域的读段，因此可以用于确定潜在未知的侧翼序列。通过汇集来自两种不同大肠杆菌的富集库。大肠杆菌菌株，并分析他们在平行，我们证明了这种方法的效率，在多重格式。至关重要的是，我们评估了大片段文库的最佳诱饵大小，并首次描述了MinION平台中目标富集的标准化方法。
The enrichment of targeted regions within complex next generation sequencing libraries commonly uses biotinylated baits to capture the desired sequences. This method results in high read coverage over the targets and their flanking regions. Oxford Nanopore Technologies recently released an USB3.0-interfaced sequencer, the MinION. To date no particular method for enriching MinION libraries has been standardized. Here, using biotinylated PCR-generated baits in a novel approach, we describe a simple and efficient way for multiplexed enrichment of MinION libraries, overcoming technical limitations related with the chemistry of the sequencing-adapters and the length of the DNA fragments. Using Phage Lambda and Escherichia coli as models we selectively enrich for specific targets, significantly increasing the corresponding read-coverage, eliminating unwanted regions. We show that by capturing genomic fragments, which contain the target sequences, we recover reads extending targeted regions and thus can be used for the determination of potentially unknown flanking sequences. By pooling enriched libraries derived from two distinct E. coli strains and analyzing them in parallel, we demonstrate the efficiency of this method in multiplexed format. Crucially we evaluated the optimal bait size for large fragment libraries and we describe for the first time a standardized method for target enrichment in MinION platform.