Amplicon Sequencing

Amplicon sequencing is a sequencing technique based on analysing specific DNA fragments (amplicons) amplified using PCR. It is most commonly used to identify microorganisms in the material under study by analysing selected genomic regions such as 16S rRNA (for bacteria), ITS (for fungi) and 18S (for eukaryotes).

Amplicon Seq workflow

Applications of Amplicon Seq

Amplicon sequencing is popular because of its precision, relatively low cost and ability to analyse many samples simultaneously.

Technical specifications of Amplicon Seq

Specifications: Illumina

• Sequencing mode: 2 x 250 bp
• Standard amount of data per sample:
  • 30k read pairs (tags)
  • 50k read pairs (tags) - standard, recommended approach
  • 100k read pairs (tags)
  • Other / Custom (available upon special request).
• Available options for selecting the amplification region:
  • 16S - bacterial identification:
    • V3+V4 - standard approach. Recommended primers: 338F and 806R
    • V4+V5 - recommended primers: 515F and 926R
    • V4 - recommended primers: 515F and 806R2
    • Archaea V3+V4 - recommended primers: Arch349F and Arch806R
  • ITS - fungal identification:
    • ITS1 - standard approach. Recommended primers: ITS1F and ITS2
    • ITS2 - Recommended primers: ITS2F and ITS2R.
  • 18S - identification of eukaryotic rRNA:
    • V4 - Recommended primers: TAReuk454FWD1 and TAReukREV3
    • V7 - Recommended primers: 960F and NSR1438

• Recommended Sample Requirements - DNA/RNA (Illumina):
  • Sample type: gDNA
  • Amount: > 500 ng
  • Concentration (NanoDrop): >20 ng/μL
  • Volume: >20 μL
  • Purity (OD260/280): 1.6 - 2.5
• MINIMUM Sample Requirements - DNA/RNA (Illumina):
  • Sample type: gDNA
  • Amount: > 300 ng
  • Concentration (Qubit): >1 ng/μL
  • Volume: >20 μL

Also available: DNA isolation from submitted samples. Details here.

Specifications: PacBio

• Long reads, HiFi
• Standard amount of data per sample to choose from:
  • 10k read pairs (tags)
  • 30k read pairs (tags)
  • 50k read pairs (tags)
  • Other / custom (available upon special request).
• Available options for selecting the amplification region:
  • 16S (the complete fragment, approximately 1.5 Kb long, from V1 to V9). Recommended primers: 27F and 1492R
  • ITS (the complete fragment, approximately 0.5-0.7 Kb long, from ITS1 to ITS2). Recommended primers: ITS1 and ITS4
  • 18S (the complete fragment, approximately 1.8 Kb long). Recommended primers: Euk-A and Euk-B
• Recommended Sample Requirements - DNA/RNA (PacBio):
  • Sample type: gDNA
  • Amount: > 1500 ng
  • Concentration (NanoDrop): >50 ng/μL
  • Volume: >20 μL
  • Purity (OD260/280): 1.6 - 2.5
• MINIMUM Sample Requirements - DNA/RNA (PacBio):
  • Sample type: gDNA
  • Amount: > 1,000 ng
  • Concentration (Qubit): >20 ng/μL
  • Volume: >20 μL

DNA extraction from submitted samples is also available. Details here.

Amplicon Seq bioinformatics analysis

The advanced bioinformatics analysis included in the Amplicon Seq package covers, among other things:

• OTU/ASV analysis
• Taxonomic annotation and relative abundance
• Alpha diversity analysis
• Beta diversity analysis
• Differential analysis between groups
• Correlation analysis
• Prediction of functional genes from 16S / ITS / 18S data