16S/18S/ITS Amplicon Sequencing-NGS
Service Advantages
● Isolation-free and rapid identification of microbial composition in environmental samples
● High resolution in low-abundant components in environmental samples
● Latest QIIME2 analyzing flow with diverse analyses in terms of database, annotation, OTU/ASV.
● High-throughput, higher accuracy
● Applicable to diverse microbial community studies
● BMK owns extensive experience with over 100,000 samples/year，covering soil, water, gas, sludge, feces, intestines, skin, fermentation broth, insects, plants, etc.
● BMKCloud facilitated data interpretation containing 45 personalized analyzing tools
Service Specifications
|
Sequencing Platform |
Library |
Recommended data yield |
Estimated turn-around time |
|
Illumina NovaSeq 6000 |
PE250 |
50K/100K/300K Tags |
30 Days |
Bioinformatics analyses
● Raw data quality control
● OTU clustering/De-noise(ASV)
● OTU annotation
● Alpha diversity
● Beta diversity
● Inter-group analysis
● Association analysis against experimental factors
● Function gene prediction
Sample Requirements and Delivery
Sample Requirements:
For DNA extracts:
|
Sample Type |
Amount |
Concentration |
Purity |
|
DNA extracts |
＞ 30 ng |
＞ 1 ng/μl |
OD260/280= 1.6-2.5 |
For environmental samples:
|
Sample type |
Recommended sampling procedure |
|
Soil |
Sampling amount: approx. 5 g; Remaining withered substance needs to be removed from surface; Grind large pieces and pass through 2 mm filter; Aliquot samples in sterile EP-tube or cyrotube for reservation. |
|
Feces |
Sampling amount: approx. 5 g; Collect and aliquot samples in sterile EP-tube or cryotube for reservation. |
|
Intestinal contents |
Samples need to be processed under aseptic condition. Wash collected tissue with PBS; Centrifuge the PBS and collect the precipitant in EP-tubes. |
|
Sludge |
Sampling amount: approx. 5 g; Collect and aliquot sludge sample in sterile EP-tube or cryotube for reservation |
|
Waterbody |
For sample with limited amount of microbial, such as tap water, well water, etc., Collect at least 1 L water and pass through 0.22 μm filter to enrich microbial on the membrane. Store the membrane in sterile tube. |
|
Skin |
Carefully scrape skin surface with sterile cotton swab or surgical blade and place it in sterile tube. |
Recommended Sample Delivery
Freeze the samples in liquid nitrogen for 3-4 hours and store in liquid nitrogen or -80 degree to long-term reservation. Sample shipping with dry-ice is required.
Service Work Flow
Sample delivery
Library construction
Sequencing
Data analysis
After-sale services
1.Species distribution
2.Heat map: Species richness clustering
3.Rare faction curve
4.NMDS analysis
5.Lefse analysis
BMK Case
Obese Individuals with and without Type 2 Diabetes show different gut microbial functional capacity and composition
Published: Cell Host & Microbe, 2019
Sequencing strategy:
Lean non-diabetes (n=633); Obese non-diabetes (n=494); Obese-Type 2 diabetes (n=153);
Target region: 16S rDNA V1-V2
Platform: Illumina Miseq (NGS-based amplicon sequencing)
Subset of DNA extracts were subjected to metagenomic sequencing on Illumina Hiseq
Key results
Microbial profilings of these metabolic diseases were successfully differentiated.
By comparing microbial features generated by 16S sequencing, obesity was found to associated with changes in microbial composition, individual features, especially significant decrease in Akkermansia, Faecalibacterium, Oscillibacter, Alistipes, etc. In addtion, T2D was found associated with increase in Escherichia/shigella.
Reference
Thingholm, L. B. , et al. “Obese Individuals with and without Type 2 Diabetes Show Different Gut Microbial Functional Capacity and Composition.” Cell Host & Microbe 26.2(2019).
