Comparative analysis of two 16S rRNA gene-based PCR primer sets provides insight into the diversity distribution patterns of anammox bacteria in different environments

Abstract

Due to the high divergence among 16S rRNA genes of anammox bacteria, different diversity pattern of the community could be resulted from using different primer set. In this study, the efficiencies and specificities of two commonly used sets, Amx368F/Amx820R and Brod541F/Amx820R, were evaluated by exploring the diversity characteristics of anammox bacteria in sediments from marine, estuary, and freshwater wetland. Statistical analysis indicated that the base mispairing rate between bases on 16S rRNA gene sequences retrieved by Amx368F/Amx820R and their corresponding ones on primer Brod541F was quite high, suggesting the different efficiency and specificity of Amx368F/Amx820R and Brod541F/Amx820R. Further experimental results demonstrated that multiple genera of anammox bacteria, including Ca. Scalindua, Ca. Brocadia, and Ca. Kuenenia, were able to be detected by Amx368F/Amx820R, but only Ca. Scalindua could be retrieved by Brod541F/Amx820R. Moreover, the phylogenetic clusters of Ca. Scalindua by Amx368F/Amx820R were different completely from those by Brod541F/Amx820R, presenting a significant complementary effect. By joint application of these two primer sets, the diversity distribution patterns of anammox bacteria in different environments were analyzed. Almost all retrieved sequences from marine sediments belonged to Ca. Scalindua. Sequences from freshwater wetland were affiliated to Ca. Brocadia and two new clusters, while high diversity of anammox bacteria was found in estuary, including Ca. Scalindua, Ca. Brocadia, and Ca. Kuenenia, corresponding to the river-sea intersection environmental feature. In total, these two prime sets have different characteristic for anammox bacteria detecting from environmental samples, and their combined application could achieve better diversity display of anammox community.

Depth-Resolved Distribution of Particle-Attached and Free-Living Bacterial Communities in the Water Column of the New Britain Trench

“…Two sets of data were generated for each of the samples, and were then combined into one dataset. Similar to previous findings ( Vahjen et al, 2010 ; Cai et al, 2013 ; Starke et al, 2014 ; Wang et al, 2015 ), the species composition of the microbial communities showed great variations between the two original datasets (Supplementary Figures S1 , S2 ). In addition, the original datasets “Qiagen-338-806” and “MP-515-907” revealed 2,438 and 1,947 OTUs, respectively.…”

“…It has been widely reported that different DNA extraction methods and primer sets targeting different hypervariable regions of 16s rRNA gene would reveal distinct species compositions of natural microbial communities ( Vahjen et al, 2010 ; Cai et al, 2013 ; Starke et al, 2014 ; Wang et al, 2015 ). In some extreme cases, dominant taxa revealed from one combination of DNA extraction and primer set can become rare taxa or even disappear in another combination ( Cai et al, 2013 ; Starke et al, 2014 ).…”

“…In some extreme cases, dominant taxa revealed from one combination of DNA extraction and primer set can become rare taxa or even disappear in another combination ( Cai et al, 2013 ; Starke et al, 2014 ). Therefore, combining data from different primer sets has been suggested to maximize the detection of microbial diversity ( Vahjen et al, 2010 ; Wang et al, 2015 ). In this study, we applied two different DNA extraction methods and two primer sets targeting different hypervariable regions of 16s rRNA gene.…”

Anaerobic Ammonium Oxidation in Acidic Red Soils

“…Candidatus Scalindua was almost the only genus found in marine sediments, such as South China Sea ( Hong et al, 2011a ), Bohai Sea ( Dang et al, 2013 ), and Arabian Sea ( Woebken et al, 2008 ). In contrast to marine environments, all of these genera of anammox bacteria have been detected in soil ecosystems ( Wang et al, 2015 ; Yang et al, 2017 ). Zhu et al (2011) found that the 16S rRNA gene sequence of fertilized paddy soil were related to four different genera of anammox bacteria.…”

Oxygen and light determine the pathways of nitrate reduction in a highly saline lake

“…systems (Yang et al, 2012;Lipsewers et al, 2016), with a totally different community structure than described in freshwater lakes (Wang et al, 2015). So far, however, very little attention has been paid to the role of anammox processes in saline lakes.…”