Analysis of 16S rRNA Amplicon Sequencing Options on the Roche/454 Next-Generation Titanium Sequencing Platform

BACKGROUND: 16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform METHODOLOGY/PRINCIPAL FINDINGS: The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management. CONCLUSIONS: Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies

Evaluating ecosystem functioning following river restoration: the role of hydromorphology, bacteria, and macroinvertebrates

Ecological restoration of freshwater ecosystems is now being implemented to mitigate anthropogenic disruption. Most emphasis is placed on assessing physico-chemical and hydromorphological properties to monitor restoration progress. However, less is known about the structural integrity and ecosystem health of aquatic ecosystems. In particular, little is known about how ecosystem function changes following river habitat restoration, especially in China. Leaf litter decomposition can be used as an indicator of stream ecosystem integrity. Therefore, the leaf breakdown rate was measured to assess the ecosystem function of restored rivers. By comparing leaf breakdown rates in urban rivers undergoing habitat restoration with that in degraded urban rivers and rivers in forested areas (i.e., reference conditions), we aimed to determine: (i) how habitat restoration affected leaf litter decomposition? (ii) the relationship between leaf litter decomposition to both environmental (habitat and physico-chemical variables) and biological factors (benthic communities), and (iii) identify the factors that contribute most to the variance in leaf litter breakdown rates. The results demonstrated a significant increase in leaf breakdown rate (120% in summer and 28% in winter) in the restored rivers compared to the degraded rivers. All environmental and biotic factors evaluated contributed synergistically to the differences in leaf litter decomposition among the three river types. The role of macroinvertebrates, mainly shredders, appeared to be particularly important, contributing 52% (summer) and 33% (winter) to the variance in decomposition, followed by habitat characteristics (e.g. substrate diversity, water velocity; 17% in summer, 29% in winter), physico-chemical variables (e.g. nutrient and organic pollutants; 11% in summer, 1% in winter) and biofilm bacteria (0% in summer, 15% in winter). Habitat restoration positively affected the structure and function of the previously degraded streams. Knowledge on controlling variables and their attribution to changes of ecosystem functioning provides guidance to assist the future planning of ecological restoration strategies