Products

16S/18S/ITS Amplicon Sequencing-PacBio

The 16S and 18S rRNA genes, along with the Internal Transcribed Spacer (ITS) region, serve as pivotal molecular fingerprinting markers due to their combination of highly conserved and hyper-variable regions, making them invaluable tools for characterizing prokaryotic and eukaryotic organisms. Amplification and sequencing of these regions offer an isolation-free approach for investigating the microbial composition and diversity across various ecosystems. While Illumina sequencing typically targets short hypervariable regions like V3-V4 of 16S and ITS1, it has been demonstrated that superior taxonomic annotation is achievable by sequencing the full length of 16S, 18S, and ITS. This comprehensive approach results in higher percentages of accurately classified sequences, achieving a level of resolution that extends to species identification. PacBio’s Single-Molecule Real-Time (SMRT) sequencing platform stands out by providing highly accurate long reads (HiFi) that cover the full-length amplicons, rivalling the precision of Illumina sequencing. This capability allows researchers to attain an unmatched advantage — a panoramic view of the genetic landscape. The extended coverage significantly elevates the resolution in species annotation, particularly within bacterial or fungal communities, enabling a deeper understanding of the intricacies of microbial populations.

Service Details

Bioinformatics

Demo Results

Featured publications

Service Features

● Sequencing platform: PacBio Revio

● Sequencing mode: CCS (HiFi reads)

● Amplification of target region followed by tandem linking of amplicons before HiFi SMRT bell library preparation

Service Advantages

● Higher taxonomic resolution: than short-amplicon sequencing, enabling higher OTU classification rates at the species level.

● Highly accurate base calling: with PacBio CCS mode sequencing (HiFi reads).

● Isolation-free: and rapid identification of microbial composition in environmental samples.

● Widely applicable: to diverse microbial community studies.

● Comprehensive bioinformatic analysis: including with the latest QIIME2 package (quantitative insight into microbial ecology) with diverse analyses in terms of database, annotation, OTU/ASV.

● Extensive Expertise: with thousands of amplicon sequencing projects conducted annually, BMKGENE brings over a decade of experience, a highly skilled analysis team, comprehensive content, and excellent post-sales support.

Service Specifications

Library	Sequencing Strategy	Data recommended	Quality control
Amplicon	PacBio Revio	10/20 K tags (read pairs)	Q30≥85%

Sample Requirements

Concentration (ng/µL)	Total amount (µg)	Volume (µL)	OD260/280
≥20	≥1	≥20	1.6-2.5

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

Previous: 16S/18S/ITS Amplicon Sequencing-NGS

Next: Assay for Transposase-Accessible Chromatin with High Throughput Sequencing (ATAC-seq)

Includes the following analysis:

●Raw data quality control

●OTU clustering/De-noise(ASV)

●OTU annotation

●Alpha diversity analysis: multiple indexes, including Shannon, Simpson and ACE.

●Beta diversity analysis

●Inter-group analysis

●Correlation analysis: between environmental factors and OUT composition and diversity

●16S functional gene prediction

Histogram of taxonomic distribution

Community distribution phylogenetic tree

Alpha diversity analysis: ACE

index

Beta diversity analysis: PCoA

Intergroup analysis: ANOVA

Explore the advancements facilitated by BMKGene’s amplicon sequencing services with PacBio through a curated collection of publications.

Gao, X. and Wang, H. (2023) ‘Comparative Analysis of Rumen Bacterial Profiles and Functions during Adaption to Different Phenology (Regreen vs. Grassy) in Alpine Merino Sheep with Two Growing Stages on an Alpine Meadow’, Fermentation, 9(1), p. 16. doi: 10.3390/FERMENTATION9010016/S1.

Li, S. et al. (2023) ‘Capturing the microbial dark matter in desert soils using culturomics-based metagenomics and high-resolution analysis’, npj Biofilms and Microbiomes 2023 9:1, 9(1), pp. 1–14. doi: 10.1038/s41522-023-00439-8.

Mu, L. et al. (2022) ‘Effects of fatty acid salts on fermentation characteristics, bacterial diversity and aerobic stability of mixed silage prepared with alfalfa, rice straw and wheat bran’, Journal of the Science of Food and Agriculture, 102(4), pp. 1475–1487. doi: 10.1002/JSFA.11482.

Yang, J. et al. (2023) ‘The Interaction between Oxidative Stress Biomarkers and Gut Microbiota in the Antioxidant Effects of Extracts from Sonchus brachyotus DC. in Oxazolone-Induced Intestinal Oxidative Stress in Adult Zebrafish’, Antioxidants 2023, Vol. 12, Page 192, 12(1), p. 192. doi: 10.3390/ANTIOX12010192.

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