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Additional file 7: Figure S6. Weighted (A) and unweighted (B) UniFrac distance-based beta diversity of the cow rumen microbial communities revealed by DNBSEQ and NovaSeq sequencing platforms.

Additional file 5: Figure S4. Comparison of the amplicon sequencing results of three typical ecological systems between DNBSEQ and NovaSeq sequencing platforms. (A) Evaluation of the reproducibility of the amplicon sequencing results of DNBSEQ and NovaSeq sequencing platform in revealing the accumulated relative abundance of microbes of abundant, moderate and rate taxa subcommunity from ecosystems with various complexity (DNB All, DNB 2, DNB 3 denotes number of all unique OTUs detected by DNBSEQ platform, consistently detected by at least 2 technical replicates, and consistently detected by all three technical replicates, respectively. Similar naming scheme was used for the NovaSeq platform). (B) Cross-verification of the OTUs identified by amplicon sequencing on DNBSEQ and NovaSeq platforms and shotgun sequencing results of the same set of mangrove sediment samples. (C) Cross-verification of the OTUs identified by amplicon sequencing on DNBSEQ and NovaSeq platforms and shotgun sequencing results of the same set of mice gut samples.

Additional file 2: Figure S1. Schematic presentation of the design and flow of this study. This study was designed to evaluate the potential harm of false positives introduced by index misassignment during the sequencing to the interpretation of metabarcoding or here amplicon sequencing studies. In part 1, mock communities with known microbial composition were used to confirm the presence of index misassignment introduced false positives and the rate of this misassignment on different sequencing platform; In part 2, samples from several typical ecosystems with various complexity were sequenced to test how index misassignment could affect the estimation of community diversity for different samples; In part 3, a case study using a set of cow rumen samples was conducted to evaluate how index misassignment caused false positives could affect the interpretation of microbial ecological roles and community assembly mechanisms.

Additional file 4: Figure S3. Venn diagrams showing the shared OTUs among distinct samples sequenced within the same batch of sequencing run on the Novaseq platform. The large number of shared OTUs among distinct samples (including two customized communities) were likely sample wise cross contaminations caused by index misassignment.

Additional file 6: Figure S5. Weighted (A) and unweighted (B) UniFrac distance-based clustering of the amplicon sequencing results revealed by DNBSEQ and NovaSeq sequencing platforms for samples from the three typical ecosystems.

Additional file 3: Figure S2. Comparison of shared fraction of OTUs by different sequencing platforms. UpSet graph showing the number of shared OTUs among the technical triplicates of DNBSEQ and NovaSeq sequencing platforms for customized mock community with four (A) or seven (B) microbes used in the current study. And heatmap showing the phylum rank relative abundances revealed by different sequencing platforms for the commercial mock (C) and customized mock communities with four (D) or seven (E) bacteria.

Additional file 8: Figure S7. Comparison of the results using unoise3 and DADA2 denoising algorithms. The accumulated OTU relative abundances in each phylum based on unoise3 and DADA2 were graphed as bar-chart side-by-side for all the mock communities and samples from three typical ecosystems.