Assessing intragenomic variation of the internal transcribed spacer two: Adapting the Illumina metagenomics protocol.
Assessing intragenomic variation of the internal transcribed spacer two: Adapting the Illumina metagenomics protocol.
复制标题
评估内部转录间隔两个的基因组内变异：调整Illumina Metagenomics方案。
DOI：
10.1371/journal.pone.0181491
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Buchheim M
中科院分区：
文献类型：
--
作者：
Alanagreh L;Pegg C;Harikumar A;Buchheim M
内转录间隔区2（ITS 2）的一级和二级结构数据已被广泛用于许多不同真核生物的多样性研究，包括绿色藻类。扩增的容易性至少部分是由于ITS 2是串联重复的rRNA阵列的一部分。基因组内变异的潜在混杂影响尚未得到解决，除了在少数生物体。此外，基因组内变异的评估很少利用合成测序方案的深度测序能力。我们目前的结果，从我们的适应16 S宏基因组测序文库制备/Illumina协议的深度测序的ITS 2基因在选定的分离株的绿色藻类属，红球藻。深度测序产生了从不到20，000到超过500，000个合并读数，超过了最近焦磷酸测序的结果。此外，这些数据的保守评估揭示了一系列的3至6个ITS 2序列单倍型（定义为独特的核苷酸多态性）在整个分类群的采样。优势单倍型的频率范围为0.35至0.98。在所有情况下，但两种情况下，具有最大频率的单倍型对应于通过使用PCR模板的桑格方法获得的序列。我们的数据还表明，合成测序方法的结果是可重复的。除了推进我们对核糖体RNA变异的理解，这项研究的结果将使我们能够开始测试关于多拷贝基因之间保持同质性的假设。
Primary and secondary structural data from the internal transcribed spacer two (ITS2) have been used extensively for diversity studies of many different eukaryotic organisms, including the green algae. Ease of amplification is due, at least in part, to the fact that ITS2 is part of the tandemly-repeated rRNA array. The potential confounding influence of intragenomic variability has yet to be addressed except in a few organisms. Moreover, few of the assessments of intragenomic variation have taken advantage of the deep sequencing capacity of sequence-by-synthesis protocols. We present results from our adaptation of the 16S Metagenomics Sequencing Library Preparation/Illumina protocol for deep sequencing of the ITS2 genes in selected isolates of the green algal genus, Haematococcus. Deep sequencing yielded from just under 20,000 to more than 500,000 merged reads, outpacing results from recent pyrosequencing efforts. Furthermore, a conservative evaluation of these data revealed a range of three to six ITS2 sequence haplotypes (defined as unique sets of nucleotide polymorphisms) across the taxon sampling. The frequency of the dominant haplotype ranged from 0.35 to 0.98. In all but two cases, the haplotype with the greatest frequency corresponded to a sequence obtained by the Sanger method using PCR templates. Our data also show that results from the sequencing-by-synthesis approach are reproducible. In addition to advancing our understanding of ribosomal RNA variation, the results of this investigation will allow us to begin testing hypotheses regarding the maintenance of homogeneity across multi-copy genes.
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