Kraková Lucia, Šoltys Katarína, Budiš Jaroslav, Grivalský Tomáš, Ďuriš František, Pangallo Domenico, Szemes Tomáš
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 84551, Bratislava, Slovakia.
Comenius University Science Park, Ilkovičova 8, 84104, Bratislava, Slovakia.
Extremophiles. 2016 Sep;20(5):795-808. doi: 10.1007/s00792-016-0855-5. Epub 2016 Jun 23.
Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1-V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing.
基于Illumina高通量DNA测序和变性梯度凝胶电泳（DGGE）-克隆技术开发了不同的方案，并将其应用于温泉相关样本的研究。该研究聚焦于三个目标基因：古菌和细菌的16S rRNA以及产甲烷微生物群落的mcrA。与桑格测序不同，Illumina目前最流行的测序技术读取长度较短，无法分析完整的16S rRNA区域或更长的基因片段。在此，我们证明无需专门针对16S rRNA短可变区设计特殊的索引或带尾引物组，因为本文提出的方法能够分析完整的细菌16S rRNA扩增子（V1-V9）以及更长的古菌16S rRNA和mcrA序列。用转座子扩增的样本由一组约300 bp长的片段组成，这些片段可通过Illumina MiSeq轻松测序。此外，观察到嵌合序列的比例较低。基于DGGE-克隆的策略通过半巢式PCR、DGGE和克隆文库构建相结合来实施。比较这两种研究方法，发现它们具有一定程度的互补性，证实DGGE-克隆方法并未过时。针对几种类型的实验室创建了新的方案，既利用了传统的DGGE技术，也采用了最现代的Illumina测序技术。
