EasyAmplicon: An easy-to-use, open-source, reproducible, and community-based pipeline for amplicon data analysis in microbiome research

Abstract

It is difficult for beginners to learn and use amplicon analysis software because there are so many software tools to choose from, and all of them need multiple steps of operation. Herein, we provide a cross-platform, open-source, and community-supported analysis pipeline EasyAmplicon. EasyAmplicon has most of the modules needed for an amplicon analysis, including data quality control, merging of paired-end reads, dereplication, clustering or denoising, chimera detection, generation of feature tables, taxonomic diversity analysis, compositional analysis, biomarker discovery, and publication-quality visualization. EasyAmplicon includes more than 30 cross-platform modules and R packages commonly used in the field. All steps of the pipeline are integrated into RStudio, which reduces learning costs, keeps the flexibility of the analysis process, and facilitates personalized analysis. The pipeline is maintained and updated by the authors and editors of WeChat official account "Meta-genome." Our team will regularly release the latest tutorials both in Chinese and English, read the feedback from users, and provide help to them in the WeChat account and GitHub. The pipeline can be deployed on various platforms, and the installation time is less than half an hour. On an ordinary laptop, the whole analysis process for dozens of samples can be completed within 3 h. The pipeline is available at GitHub (https://github.com/YongxinLiu/EasyAmplicon) and Gitee (https://gitee.com/YongxinLiu/EasyAmplicon).

Keywords: amplicon; bioinformatics; metagenome; microbiome; pipeline; visualization.

Figure 1

Pipeline of EasyAmplicon for analyzing paired‐end amplicon sequences. (A) Dimensionality reduction: processing raw sequencing reads into feature tables. (B) Analysis: providing phylogenetic analysis, taxonomic classification, functional prediction, and alpha‐ and beta‐diversity calculations. (C) Statistics and visualization: generating publication‐quality figures and performing statistical tests for biological interpretations. ASVs, amplicon sequence variants; OTU, operational taxonomic units.

Figure 2

Examples of publication‐quality visualizations. (A) Boxplot showing alpha diversity in richness metrics among groups. Different letters indicate statistical significance among groups (p < 0.05, ANOVA, Tukey HSD). The horizontal bars within boxes represent medians. The tops and bottoms of the boxes represent the 75th and 25th percentiles, respectively. The upper and lower whiskers extend to data no more than 1.5× the interquartile range from the upper and lower edge of the box, respectively. (B) Rarefaction curve of richness shows that features reach saturation stage with increasing sequencing depth. Each vertical bar represents standard error. (C) Heatmap based on Bray−Curtis dissimilarity. (D) Principal coordinate analysis (PCoA) of Bray−Curtis dissimilarity. (E) Stacked bar plot of taxonomic composition in grouped samples at phylum level. (F) Tree map of taxonomic composition. (G) Volcano plot showing significantly differential abundance taxa between KO and WT groups. (H) Manhattan plot showing different features and related taxa between KO and WT groups. The numbers of replicated samples in this figure are as follows: in KO (n = 6), OE (n = 6), and WT (n = 6). KO, knock‐out; OE, overexpression; WT, wild‐type.

Figure 3

Supplementary examples of publication‐quality visualizations to Figure 2. (A) Venn diagram showing common and unique ASVs (relative abundance >0.1%) among three groups. (B) Constrained principal coordinate analysis (CPCoA) of three groups. (C) Stacked plot of average relative abundance at phylum level of three groups. (D) Circle plot of average relative abundance at phylum level of three groups. (E) Heatmap showing significantly different ASVs between KO and WT groups (Wilcoxon test, p < 0.05). ASVs, amplicon sequence variants; KO, knock‐out; WT, wild‐type.

Figure 4

Visualizations generated by third‐party software using the intermediate files of EasyAmplicon. (A) Extended error bar plot at genus level in WT and KO groups by STAMP. (B) Cladogram showing biomarkers in each group by LEfSe. (C) Percentage of BugBase annotated anaerobic bacteria at the phylum level. (D) Phylogenetic tree of 86 ASVs (relative abundance > 0.2%). The tree background is colored by Phylum. The outer strip represents different classes. The heatmap represents the average relative abundance of all samples. The bar plot represents the relative abundance of the WT group. ASVs, amplicon sequence variants; KO, knock‐out; WT, wild‐type.