Amplicon Sequencing

Technical Specifications

TECHNICAL SPECIFICATIONS

Item	Details
Sequencing Platform	Illumina MiSeq PE250（16S / 18S / ITS）
Analysis Strategy	OTU / ASV analysis, 50,000 reads per sample
Taxonomy Databases	RDP classifier Bayesian algorithm; reference databases Silva 138 (16S/18S) and Unite 7.0 (ITS); community composition statistics across 7 ranks (domain to species)
Functional Prediction	PICRUSt2 predicts functional abundance from marker genes (KEGG orthologs KO, EC enzyme classes)
Standard Delivery	Raw data + analysis report

Analysis Workflow

ANALYSIS WORKFLOW

Data QC

PE merging · filtering

OTU Clustering

Dereplication · chimera removal

Taxonomy Annotation

RDP · Silva 138

Composition

Venn · Bar · heatmap

Alpha Diversity

Rarefaction · indices

Beta Diversity

PCoA · NMDS

Differential Taxa

LEfSe · rank-sum tests

Functional Prediction

PICRUSt2 · KEGG

Bioinformatics Analyses

BIOINFORMATIC MODULES

Module	Core Methods & Metrics	Key Outputs
Data QC	PE reads merged by overlap; sequences are quality-controlled, filtered and demultiplexed before clustering/denoising and taxonomic analysis	Filtering statistics table, base-quality distribution plots
OTU Clustering	Non-redundant sequences extracted and singletons removed; OTUs clustered at 97% similarity with chimera removal during clustering	OTU abundance table, annotated OTU table, taxon statistics table
Taxonomy Annotation	Taxonomic annotation with the RDP classifier Bayesian algorithm; Silva 138 (16S/18S) and Unite 7.0 (ITS) reference databases; composition statistics across 7 ranks (domain/kingdom/phylum/class/order/family/genus/species)	Absolute / relative abundance tables at each taxonomic rank
Composition	Shared and unique OTU statistics between groups; community bar plots at each taxonomic level; abundance heatmap with two-way clustering of taxa and samples	Venn diagram, community bar plots, heatmap
Alpha Diversity	Richness (Chao1/Ace), diversity (Shannon/Simpson), evenness (Pielou), phylogenetic diversity (PD) and coverage; rarefaction, Shannon-Wiener and rank-abundance curves	Three curve plots, intergroup boxplots of indices and test tables
Beta Diversity	Inter-sample distance matrices (Bray-Curtis / Jaccard / UniFrac); UPGMA hierarchical clustering; PCA / PCoA / NMDS ordination; ANOSIM / adonis intergroup tests	Distance-matrix heatmap, clustering tree, PCA / PCoA / NMDS plots
Differential Taxa Analysis	LEfSe linear discriminant analysis effect size (LDA scores); Wilcoxon rank-sum test for two groups and Kruskal test for multiple groups to identify significantly enriched taxa	LEfSe cladogram, LDA bar chart, differential test tables
Functional Prediction	PICRUSt2 prediction of functional abundance from marker genes (KEGG orthologs KO, EC classes); PCoA of functional units; LEfSe and MetagenomeSeq differential analysis of KEGG / MetaCyc pathways	Functional abundance tables, functional PCoA plot, pathway LEfSe plots

Example Results

SELECTED FIGURES FROM ACTUAL REPORTS

Shared / Unique OTU Venn Diagram

Shared and unique OTUs across KO / OE / WT groups; numbers in overlapping areas indicate shared features.

Community Composition Bar Plot (Genus Level)

Relative genus-level abundance per sample; low-abundance taxa grouped as 'other'.

Taxon Abundance Heatmap (Genus Level)

Abundance distribution of top genera across samples, with two-way hierarchical clustering of taxa and samples.

Rarefaction Curve

Observed taxa versus randomly sampled sequences, assessing whether sequencing depth is sufficient.

Shannon-Wiener Curve

Shannon diversity index at increasing sequencing depths; a plateau indicates sufficient data volume.

Alpha Diversity Intergroup Boxplot

Distribution and significance testing of the Ace richness index across groups, with significance markers.

PCA (OTU Level)

Principal-component score plot of community-structure differences, annotated with R² and test p-values.

PCoA (Bray-Curtis)

Principal coordinates analysis on distance matrices; confidence ellipses show intergroup structural differences.

LEfSe Cladogram

Taxonomic distribution of significantly different taxa between groups; colors indicate the enriched group.

Applications

APPLICATIONS

Gut Microbiota ProfilingEnvironmental Microbial DiversityFermentation Microbiome MonitoringPlant Rhizosphere MicrobiomeMicrobiome-Metabolome Integration

FAQ

How long does a Amplicon Sequencing project take?

From sample QC approval, standard timelines are 3-6 weeks (library prep, sequencing and bioinformatics included); large cohorts or deep custom analyses follow the confirmed study plan.

What if my samples fail QC?

Failed QC is reported immediately with reasons, and we discuss resampling or plan adjustments with you — sequencing never proceeds without qualification.

What bioinformatics is included? Can I add custom analyses?

Standard delivery covers QC, mapping/assembly, quantification, differential and functional enrichment analyses. Custom deep-dive and multi-omics integrative analyses are supported on request.

What samples do you need for Amplicon Sequencing?

Total RNA ≥1 μg with RIN ≥7 (snap-frozen, dry ice); microbial and faecal samples ≥200 mg (sterile, −80 °C); whole blood for WGS ≥500 μL (EDTA only, never heparin). RNA integrity matters more than concentration. See Technical Resources → Sample Guide.