Full-length Amplicon Sequencing

16S/18S/ITS Amplicon sequencing

Faster, Accurate, Affordable
Analyse in any way you like!

As a leading provider of genomic services, BMK offers diverse genomic solutions to meet different research goals in microbial diversity, taxonomy and phylogeny studies.  We are equipped with all the cutting-edge sequencing platforms, covering NGS and NNGS technologies. Our expert team owns massive experience on amplicon sequencing of over 4,000 projects, generating over 65 high impact publications. In addition, we are excited to offer our clients “full-length amplicon sequencing” with PacBio sequencing platform, which achieves an annotation resolution at species-level. Our own-developed bioinformatic analysis platform, BMKCloud, enables our clients to have more flexible parameter design and process multiple analysis in parallel. On BMKCloud, you can analyse you data in any way you like! 

What 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


11

Identification and abundancy


Species distribution histogram Species richness heatmap phylogenetic tree ...
22

Differences


Alpha diversity Beta diversity Lefse analysis Random forest ...
33

Functions


KEGG function prediction COG function prediction ...
44

Correlations


RDA/CCA analysis Species correlation network Correlation heatmap ...

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

Tired of the EXTREMLY LONG Email thread to get the analysis you want?


With BMKCloud
0 bioinformatic knowledge needed
Analyse your data as you like


What is BMKCloud?


BMKCloud is a visual bioinformatics analysis platform with a collection of  all commonly used tools for data analysis, including raw data processing, assembly, annotation, statistics, correlation analysis, etc. Here, to obtain your ideal analysis, you only need to know what you want. From raw data to figures ready for publicating, only several clicks are needed. No programming background or bioinformatic knowledge is required. Additionally, almost all parameters are open for customized setting to meet your diverse research goals.






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. 

Free for Grouping Design
Free for Parameter Setting


Once data is ready, all figures and analysis are open for group settings to meet diverse research goals. Multiple group settings are available to be analysed in parallel.
Almost all parameters in analysis are free for customized setting in case of specific requirements, such as classification level, mathematic model for statistic analysis, threshold for significancy, etc. Don't worry if you are not familiar with all parameters, BMKCloud provides brief explanation and default settings for all crucial parameters based on common research needs. And our highly experienced team are more than happy to help you more.

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