Diversity analysis of sulfite- and sulfate-reducing microorganisms by multiplex dsrA and dsrB amplicon sequencing using new primers and mock community-optimized bioinformatics
Diversity analysis of sulfite- and sulfate-reducing microorganisms by multiplex dsrA and dsrB amplicon sequencing using new primers and mock community-optimized bioinformatics
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DOI：
10.1111/1462-2920.13139
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发表时间：
2016-09-01
影响因子：
5.1
通讯作者：
Loy, Alexander
中科院分区：
文献类型：
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作者：
Pelikan, Claus;Herbold, Craig W.;Loy, Alexander
异化亚硫酸盐还原酶（DsrAB）基因在亚硫酸盐和硫酸盐还原微生物的生态学研究中常被用作诊断标记。在这里，我们开发了新的高覆盖率引物组，用于生成还原性细菌型dsrA和dsrB聚合酶链反应（PCR）产物，用于高度平行的扩增子测序和生物信息学工作流程，用于短dsrA和dsrB读数的处理和分类。我们采用了两个不同的模拟社区，由45或90个已知的dsrAB序列来源于环境克隆，以精确地评估我们的扩增子测序方法的各个步骤在Illumina MiSeq平台上的性能。虽然PCR循环数，基因特异性引物错配和高质量序列的严格过滤观察到的dsrA和dsrB社区结构有显着的影响，大多数模拟社区序列的恢复一般是成比例的相对输入丰度。在选定的环境样品中成功的dsrA和dsrB多样性分析进一步证明了多重扩增子测序方法足以监测含dsrAB微生物的空间分布和时间丰度动态。虽然测试还原细菌型dsrAB，这种方法是很容易适用于氧化型dsrAB的硫氧化细菌，也提供了指导处理其他功能基因的短扩增子读取。
Genes encoding dissimilatory sulfite reductase (DsrAB) are commonly used as diagnostic markers in ecological studies of sulfite- and sulfate-reducing microorganisms. Here, we developed new high-coverage primer sets for generation of reductive bacterial-type dsrA and dsrB polymerase chain reaction (PCR) products for highly parallel amplicon sequencing and a bioinformatics workflow for processing and taxonomic classification of short dsrA and dsrB reads. We employed two diverse mock communities that consisted of 45 or 90 known dsrAB sequences derived from environmental clones to precisely evaluate the performance of individual steps of our amplicon sequencing approach on the Illumina MiSeq platform. Although PCR cycle number, gene-specific primer mismatches and stringent filtering for high-quality sequences had notable effects on the observed dsrA and dsrB community structures, recovery of most mock community sequences was generally proportional to their relative input abundances. Successful dsrA and dsrB diversity analysis in selected environmental samples further proved that the multiplex amplicon sequencing approach is adequate for monitoring spatial distribution and temporal abundance dynamics of dsrAB-containing microorganisms. Although tested for reductive bacterial-type dsrAB, this method is readily applicable for oxidative-type dsrAB of sulfur-oxidizing bacteria and also provides guidance for processing short amplicon reads of other functional genes.