16S/18S/ITS Amplicon Sequencing-PacBio
Service Advantages
● Long-reads revealing full-length sequence of 16S/18S/ITS
● Highly accurate base calling with PacBio CCS mode sequencing
● Species-level resolution in OTU/ASV annotation
● Latest QIIME2 analyzing flow with diverse analyses in terms of database, annotation, OTU/ASV.
● 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 |
Turnaround Time |
|
PacBio Sequel II |
SMRT-bell |
5K/10K/20K Tags |
44 Working 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 |
＞ 1 μg |
＞ 20 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.Annotation rate of V3+V4(Illumina)-based microbial community profiling vs. Full-length (PacBio)-based profiling.
(Data of 30 randomly picked projects were applied for statistics)
2.Annotation rate of full-length amplicon sequencing at species-level in different sample types
3.Species distribution
4.Phylogenetic tree
BMK Case
Arsenic exposure induces intestinal barrier damage and consequent activation of gut-liver axis leading to inflammation and pyroptosis of liver in ducks
Published: Science of the Total Environment, 2021
Sequencing strategy:
Samples: Control vs 8 mg/kg ATO exposed group
Sequencing data yield: 102,583 raw CCS sequences in total
Control: 54,518 ± 747 effective CCS
ATO-exposed : 45,050 ± 1675 effective CCS
Key results
Alpha diversity: ATO exposure significantly altered the intestinal microbial richness and diversity in the ducks.
Metastats analysis:
In phylum level: 2 bacterial phyla only detected in control groups
In genus level: 6 genera were found significantly different in relative abundance
In species level: 36 species were identified in total, with 6 of them significantly different in relavive abundance
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).
