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ABSTRACT Targeted amplicon sequencing is widely used in microbial ecology studies. However, sequencing artifacts and amplification biases are of great concern. To identify sources of these artifacts, a systematic analysis was performed using mock communities comprised of 16S rRNA genes from 33 bacterial strains. Our results indicated that while sequencing errors were generally isolated to low-abundance operational taxonomic units, chimeric sequences were a major source of artifacts. Singleton and doubleton sequences were primarily chimeras. Formation of chimeric sequences was significantly correlated with the GC content of the targeted sequences. Low-GC-content mock community members exhibited lower rates of chimeric sequence formation. GC content also had a large impact on sequence recovery. The quantitative capacity was notably limited, with substantial recovery variations and weak correlation between anticipated and observed strain abundances. The mock community strains with higher GC content had higher recovery rates than strains with lower GC content. Amplification bias was also observed due to the differences in primer affinity. A two-step PCR strategy reduced the number of chimeric sequences by half. In addition, comparative analyses based on the mock communities showed that several widely used sequence processing pipelines/methods, including DADA2, Deblur, UCLUST, UNOISE, and UPARSE, had different advantages and disadvantages in artifact removal and rare species detection. These results are important for improving sequencing quality and reliability and developing new algorithms to process targeted amplicon sequences. IMPORTANCEAmplicon sequencing of targeted genes is the predominant approach to estimate the membership and structure of microbial communities. However, accurate reconstruction of community composition is difficult due to sequencing errors, and other methodological biases and effective approaches to overcome these challenges are essential. Using a mock community of 33 phylogenetically diverse strains, this study evaluated the effect of GC content on sequencing results and tested different approaches to improve overall sequencing accuracy while characterizing the pros and cons of popular amplicon sequence data processing approaches. The sequencing results from this study can serve as a benchmarking data set for future algorithmic improvements. Furthermore, the new insights on sequencing error, chimera formation, and GC bias from this study will help enhance the quality of amplicon sequencing studies and support the development of new data analysis approaches. 

ABSTRACT Tundra ecosystems are typically carbon (C) rich but nitrogen (N) limited. Since biological N 2 fixation is the major source of biologically available N, the soil N 2 -fixing (i.e., diazotrophic) community serves as an essential N supplier to the tundra ecosystem. Recent climate warming has induced deeper permafrost thaw and adversely affected C sequestration, which is modulated by N availability. Therefore, it is crucial to examine the responses of diazotrophic communities to warming across the depths of tundra soils. Herein, we carried out one of the deepest sequencing efforts of nitrogenase gene ( nifH ) to investigate how 5 years of experimental winter warming affects Alaskan soil diazotrophic community composition and abundance spanning both the organic and mineral layers. Although soil depth had a stronger influence on diazotrophic community composition than warming, warming significantly ( P <  0.05) enhanced diazotrophic abundance by 86.3% and aboveground plant biomass by 25.2%. Diazotrophic composition in the middle and lower organic layers, detected by nifH sequencing and a microarray-based tool (GeoChip), was markedly altered, with an increase of α-diversity. Changes in diazotrophic abundance and composition significantly correlated with soil moisture, soil thaw duration, and plant biomass, as shown by structural equation modeling analyses. Therefore, more abundant diazotrophic communities induced by warming may potentially serve as an important mechanism for supplementing biologically available N in this tundra ecosystem. IMPORTANCE With the likelihood that changes in global climate will adversely affect the soil C reservoir in the northern circumpolar permafrost zone, an understanding of the potential role of diazotrophic communities in enhancing biological N 2 fixation, which constrains both plant production and microbial decomposition in tundra soils, is important in elucidating the responses of soil microbial communities to global climate change. A recent study showed that the composition of the diazotrophic community in a tundra soil exhibited no change under a short-term (1.5-year) winter warming experiment. However, it remains crucial to examine whether the lack of diazotrophic community responses to warming is persistent over a longer time period as a possibly important mechanism in stabilizing tundra soil C. Through a detailed characterization of the effects of winter warming on diazotrophic communities, we showed that a long-term (5-year) winter warming substantially enhanced diazotrophic abundance and altered community composition, though soil depth had a stronger influence on diazotrophic community composition than warming. These changes were best explained by changes in soil moisture, soil thaw duration, and plant biomass. These results provide crucial insights into the potential factors that may impact future C and N availability in tundra regions.