Dataset Information

Reproducibility and quantitation of amplicon sequencing-based detection.

ABSTRACT: To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating ?-diversity but less on ?-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative analysis of the ?-diversity of microbial communities.

SUBMITTER: Zhou J

PROVIDER: S-EPMC3146266 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Reproducibility and quantitation of amplicon sequencing-based detection.

Zhou Jizhong J Wu Liyou L Deng Ye Y Zhi Xiaoyang X Jiang Yi-Huei YH Tu Qichao Q Xie Jianping J Van Nostrand Joy D JD He Zhili Z Yang Yunfeng Y

The ISME journal 20110224 8

To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The techni ...[more]

PMID: 21346791

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Project description:Illumina's MiSeq has become the dominant platform for gene amplicon sequencing in microbial ecology studies; however, various technical concerns, such as reproducibility, still exist. To assess reproducibility, 16S rRNA gene amplicons from 18 soil samples of a reciprocal transplantation experiment were sequenced on an Illumina MiSeq. The V4 region of 16S rRNA gene from each sample was sequenced in triplicate with each replicate having a unique barcode. The average OTU overlap, without considering sequence abundance, at a rarefaction level of 10,323 sequences was 33.4±2.1% and 20.2±1.7% between two and among three technical replicates, respectively. When OTU sequence abundance was considered, the average sequence abundance weighted OTU overlap was 85.6±1.6% and 81.2±2.1% for two and three replicates, respectively. Removing singletons significantly increased the overlap for both (~1-3%, p<0.001). Increasing the sequencing depth to 160,000 reads by deep sequencing increased OTU overlap both when sequence abundance was considered (95%) and when not (44%). However, if singletons were not removed the overlap between two technical replicates (not considering sequence abundance) plateaus at 39% with 30,000 sequences. Diversity measures were not affected by the low overlap as α-diversities were similar among technical replicates while β-diversities (Bray-Curtis) were much smaller among technical replicates than among treatment replicates (e.g., 0.269 vs. 0.374). Higher diversity coverage, but lower OTU overlap, was observed when replicates were sequenced in separate runs. Detrended correspondence analysis indicated that while there was considerable variation among technical replicates, the reproducibility was sufficient for detecting treatment effects for the samples examined. These results suggest that although there is variation among technical replicates, amplicon sequencing on MiSeq is useful for analyzing microbial community structure if used appropriately and with caution. For example, including technical replicates, removing spurious sequences and unrepresentative OTUs, using a clustering method with a high stringency for OTU generation, estimating treatment effects at higher taxonomic levels, and adapting the unique molecular identifier (UMI) and other newly developed methods to lower PCR and sequencing error and to identify true low abundance rare species all can increase reproducibility.

Project description:Benefiting from the rapid developments and wide applications of high-throughput sequencing, great advancements have been made in investigating microbiota, which are highly diverse and play key roles in both element cycling and the energy flow of ecosystems. There have been inherent limitations of amplicon sequencing that could introduce uncertainty and raise concerns about the accuracy and reproducibility of this technology. However, studies focusing on the reproducibility of amplicon sequencing are limited, especially in characterizing microbial communities in deep-sea sediments. To evaluate reproducibility, 118 deep-sea sediment samples were used for 16S rRNA gene sequencing in technical replicates (repeated measurements of the same sample) that demonstrate the variability of amplicon sequencing. The average occurrence-based overlaps were 35.98% and 27.02% between two and among three technical replicates, respectively, whereas their abundance-based overlaps reached 84.88% and 83.16%, respectively. Although variations of alpha and beta diversity indices were found between/among technical replicates, alpha diversity indices were similar across samples, and the average beta diversity indices were much smaller for technical replicates than among samples. Moreover, clustering methods (i.e., operational taxonomic units [OTUs] and amplicon sequence variants [ASVs]) were shown to have little impact on the alpha and beta diversity patterns of microbial communities. Taken together, although there are variations between/among technical replicates, amplicon sequencing is still a powerful tool with which to reveal diversity patterns of microbiota in deep-sea sediments. IMPORTANCE The reproducibility of amplicon sequencing is vital for whether the diversities of microbial communities could be accurately estimated. Thus, reproducibility influences the drawing of sound ecological conclusions. Nevertheless, few studies have focused on the reproducibility of microbial communities that are characterized by amplicon sequencing, and studies focusing on microbiota in deep-sea sediments have been especially lacking. In this study, we evaluated the reproducibility of amplicon sequencing targeting microbiota in deep-sea sediments of cold seep. Our results revealed that there were variations between/among technical replicates and that amplicon sequencing was still a powerful tool with which to characterize the diversities of microbial communities in deep-sea sediments. This study provides valuable guidelines for the reproducibility evaluation of future work in experimental design and interpretation.

Dataset Information

Reproducibility and quantitation of amplicon sequencing-based detection.

Publications

Reproducibility and quantitation of amplicon sequencing-based detection.

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