16S/18S/ITS Amplicon sequencing
Third-generation SequencingWhat is 16S/18S/ITS amplicon sequencing?
Microbial community mapping has long been limited by the experimental difficulties in traditional methodologies at cell levels, until sequencing technology comes onto the stage with growing throughput and shrinking cost. The subunit on 16S rRNA containing both highly conserved and hypervariable regions (Figure 1a.), firstly discovered in 1977 by Woese et al., is a perfect molecular fingerprint for prokaryotic organisms identification. Subsequently, similar structures on 18S rRNA (Figure 1b.) were described and applied for eukaryotes identification.Taking advantage of sequencing, these amplicons can be targeted based on the conserved parts and the hypervariable regions can be fully charaterised for microbial identification contributing to studies covering microbial diversity analysis, taxonomy, phylogeny, etc.


Application Region
Human: mouth, skin, feces, intestines...
Animals: rumen, intestines...
Plant: mycorrhiza, endophytes...
Soil: grassland, mine, glaciers, frozen soil...
Water: sea water, river water...
Industry: fermentation broth, koji...
Insects: mosquitoes, mites...
Environment: mine, volcano, air...
What we can do?
NGS 16S/18S/ITS amplicon sequencing workflow


Identification and abundancy

Differences

Functions

Correlations
Full-length amplicon sequencing -- WHAT'S NEW?
To data, Pacific Bioscience (PacBio) has successfully extended the reading of sequences to tens of kilobases. Breaking the limitations of short reads, the new platforms (Fig. 2a) allow us to obtain full-length reads of 16s rRNA (1,000 bp-1,500 bp) in bacteria or 18S rRNA (1,500 bp-2,000 bp) and ITS regions (400 bp-900 bp) in eukaryotics. The widen view of genetic field greatly enhanced the resolution of species annotation and functional genes. The long concerned issue on the accuracy of third-generation sequencing has been largely improved by construction of circular consensus sequences (Fig. 2b).
Project workflow
Bioinformatics workflow


Project Advantages
Long reads revealing full-length sequence of 16S/18S/ITS
In short-read sequencing platforms, the size of targets are typically restricted to 100 to 500 bp, where the information provided is largely limited to fragments of taxonomic barcodes, such as v3, v4 regions on 16S rRNA, ITS1 or ITS2, etc. Attribute to the development in third-generation sequencing, long reads that could cover the entire preferable regions for identification (ca. 1.5-2.0 kb) without reduction in accuracy are available by applying PacBio sequencing system in amplicon sequencing studies. The sequence obtained from NGS and PacBio sequencing system are shown in Fig. 4.

Comparable base accuracy
PacBio CCS library allows multiple rounds of sequencing around the library to perform self-correction. In our cases, CCS mode can reach 5 passes at minimum to largely ensure the accuracy of reading. According to PacBio announced data, single read accuracy can reach 99% after 4 passes.Achieving “species-level” resolution
Taking advantages of both long reads and high-throughput, the accuracy of annotation can be dramatically increased since genius-level and reach an average species-level annotation rate of 60% (Fig. 5)
Project |
Platform |
Annotation rate at genius |
Annotation rate at species |
NGS amplicon sequencing |
HiSeq2500 |
78% |
6% |
Full-length amplicon sequencing |
PacBio |
95% |
60% |
Project specification
Sample types: Water, sludge, soil, gut, feces, human source samples as well as DNA or PCR products, etc.
Typical turnaround time: approximately 45 days.
Data saturation: In general, 5,000 CSS allows identification of dominant flora. 10,000 CSS starts reaching OTU discovery saturation. 20,000 CSS reaches almost full saturation in OTU numbers. (Saturation curve of some typical sample types are shown in Fig 5.)
Annotation rate: Average annotation rate of typical sample types at species-level are shown in the table below.

Sample Type |
Feces |
Gut |
Soil |
Water |
Average rate of annotation at species |
64.7% |
69.4% |
61.7% |
68.4 |
What is BMKCloud?
Clear user interface
BMKCloud provides our clients an extremely clear and user-friendly interface to process their customized analysis in no time.
Simple, Direct, Ease of learning

Free customized parameter settings
On BMKCloud our clients are free to design any analysis on sequencing data and generate figures fulfill their specific demand. On BMKCloud, YOU control and initiate all actions and the platform do the rest for you.


Comprehensive analysis
BMKClouds provides 30+ items of basic and advanced analysis, including taxonomic analysis, alpha diversity, beta diversity, Lefse analysis, functional genes prediction, statistical analysis, which covers almost all aspects of community studies.

BMKCloud also offers correlation analysing tools enabling our clients to analyse correlations between samples or groups, including RDA/CCA analysis, db-RDA analysis, Mantel Test analysis, Correlation heatmap and Correlation network. Moreover, customized environmental factors can also be uploaded here in order to connect species information of samples with environmental factors.

Rapid analysis
Compared to traditional service model, there are NO intermediate steps needed during bioinformatic analysis part. With BMKCloud, you are completely released from the time-consuming and error-prone steps of clarifying specific analysing demand to the technical support team, then to bioinformatics teams. All you need to do is simply several clicks on the platform. It only takes in average of ONE or TWO HOURs to complete the analysis report of your own preference.

24-hour Real-time projects follow-up and problem solving
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