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Microbial Community Analysis

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Overview

Using genomic DNA extracted from samples such as feces or soil, we perform PCR amplification of marker genes such as 16S rRNA, followed by deep sequencing. This process identifies the types and relative abundances of microbes present in the samples.

Workflow

 

Genomic DNA Extraction: From soil, feces, etc.
↓
1st PCR: Amplify target regions and add linker sequences.
↓
2nd PCR: Add adapter sequences.
↓
Sequencing
↓
Standard Data Analysis: Includes microbial composition and α-diversity calculations.
↓ ↓ ↓
PCoA Plot Statistical Significance Testing
(PERMANOVA)		 Statistical Significance Testing
(Differential Abundance Analysis)

Target Regions (PCR Primers)

We provide the following primers as standard.
Please contact us if other target regions are required.

Target Region Primer Features Reference
Bacteria 16S rRNA V3-V4
(341F / 805R)		 Our standard primer for amplifying bacteria and some archaea. Herlemann, Daniel PR, et al. The ISME
journal 5.10 (2011): 1571-1579.
Bacteria 16S rRNA V3-V4
(341F’ / 805R)		 Improved efficiency for archaea compared to 341F / 805R. Hugerth, Luisa W., et al. Applied and
environmental microbiology 80.16
(2014): 5116-5123.
Bacteria 16S rRNA V3-V4
(335F / 769R)		 Suppresses amplification of plant chloroplast 16S rRNA, ideal for plant-contaminated samples. Dorn-In, Samart, et al. Journal of
microbiological methods 113 (2015):
50-56.
Fungi ITS1
(ITS1-F_KYO1 / ITS2_KYO2)		 Primer set for fungi. Toju, Hirokazu, et al. PloS one 7.7
(2012): e40863.

Analysis Plan

Shared Sequencing

Data Amount: MiSeq 300bp paired-end sequencing (~100,000 read pairs/sample).

Pricing (excl. tax):
DNA extraction 15,000 JPY / sample or conducted by the customer.*
1st PCR & purification 10,000JPY / sample or conducted by the customer.
2nd PCR & purification 60,000 JPY / sample.
MiSeq sequencing
Data analysis
* The price for DNA extraction may vary depending on the type, quantity, and form of the sample.

Dedicated Run

Data Amount: MiSeq 300bp paired-end sequencing (~16 million read pairs/run, ~30% PhiX control).

* Contact us for pricing and turnaround time.

Sample Requirements

● If Providing Samples for DNA Extraction
Sample Type Required Amount Storage/Shipping Temperature
Sludge 5 – 10 mL Refrigerated
Soil 1 – 5 g Refrigerated
Feces 0.2 – 0.5 g Frozen
Saliva 0.2 – 1 mL Frozen
* Please contact us for samples outside the above categories.
● If Providing Genomic DNA
Sample Type Total Amount Concentration Volume
Purified Genomic DNA ≥ 150 ng ≥ 5 ng/µL ≥ 30 µL
* Contact us if your sample does not meet these requirements.
● If Providing 1st PCR Products
Sample Type Total Amount Concentration Volume
Purified 1st PCR Product ≥ 5 ng ≥ 0.2 ng/µL ≥ 20 µL
* Contact us if your sample does not meet these requirements.
* Please use the 1st PCR primers provided by us or primers with our specified linker sequences.
● If Providing 2nd PCR Products
Sample Type Total Amount Concentration Volume
Purified 2nd PCR Product ≥ 150 ng ≥ 5 ng/µL ≥ 20 µL
* Contact us if your sample does not meet these requirements.
* Shared sequencing service is not available. A full MiSeq 1RUN is required.

Standard Data Analysis

【Microbial Composition Calculation】

Identifies the types and relative abundances of microbes.

[Microbial composition]

【α-Diversity Calculation】

Provides indicators of microbial diversity and data sufficiency.

[α-Diversity]

【PCoA Plot】

Visualizes similarities between samples based on microbial composition.

[PCoA Plot]

【Statistical Significance Testing (PERMANOVA)】

Tests for significant differences in microbial composition between specified groups.

[PCoA image]

Optional Data Analysis

【Statistical Significance Testing (Differential Abundance Analysis)】

Examines the statistical significance of differences in microbial abundances between groups.

[Differential Abundance Analysis]

【Integration of Multiple Datasets】

Re-analyzes and integrates datasets from multiple analysis runs.

[Integration of multiple datasets]

Frequently Asked Questions

Q: How much sequencing data is required for analysis?

The required data amount varies depending on microbial diversity, but our standard plan includes ~100,000 read pairs/sample.
α-diversity analysis can help confirm data sufficiency.

Q: What are the differences between QIIME and QIIME2?

QIIME generates representative sequences (Operational Taxonomy Unit: OTU), which may include sequences from closely related species.
QIIME2, by contrast, offers more precise error correction and species-level resolution.

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