Sequencing the bacterial 16S gene is a common method for identifying microbes and their relative abundance from complex samples with high diversity. 16S microbiome profiling uses PCR to target the DNA sequence of the 16S ribosomal RNA (rRNA) gene which is found in all microbes.
Current 16S sequencing techniques fall short:
- Partial 16S sequencing is unable to differentiate between physiologically different phyla
- Full length 16S rRNA sequences can be identical in many similar bacterial species
- Shotgun metagenomic sequencing can be expensive and computationally onerous
- Some samples are difficult to analyze due to non-bacterial host DNA drowning out microbes of interest
The better solution: Actionable 16S-ITS-23S NGS Analysis with SBanalyzer™ and Athena™ Microbial Reference Database
Long read amplicons enable high accuracy, cost-effective microbiome analysis of complex samples. Combining long-read NGS with sequencing beyond the 16S to include the more heterogenous internal transcribed spacer (ITS) region dramatically improves the ability distinguish bacterial strains of physiological importance. Shoreline Biome’s long-read microbiome assays provide an improved sample prep and mapping for full-length 16S but also provide deeper taxonomic resolution by leveraging a novel 2500 base pair amplicon containing the entire 16S rRNA gene as well as the variable ITS region and a segment of the 23S gene.
Shoreline Biome Kits Enable:
- Fast and easy preparation of HMW ssDNA from microbe containing samples
- One-step PCR assay to generate dual ended 16S-ITS-23S amplicons or full-length 16S amplicons
- Multiplex amplicon pools of 10s to 1000s* of samples
*depending on sequencer and type of samples
- Easily demultiplex and map to the highest taxonomic resolution possible using the complimentary SBanalzyer™ software with embedded long read Athena™ Microbial Reference Database.
Available for PacBio Sequel and Oxford Nanopore Technologies NGS platforms
Not all microbial databases are created equal
Maximize Resolution Using SBanalyzer Powered by the Athena Microbial Reference Database
Traditional 16S rRNA analysis software developed for short read analysis do not include the important sequences contained within the ITS region, essential for high resolution taxonomic assignment. The Athena database is a one-of -a-kind microbial database that addresses both the similarities between 16S regions as well as the highly variable ITS region. SBanalyzer and Athena Microbial Reference Database, combined with Shoreline Biome’s long amplicon prep kits enables the discovery of never before detected species and strains from microbiome samples. This easily downloadable and executable software tool simplifies and improves taxonomic assignment from the Shoreline Biome StrainID, NanoID and V1-V9 kits by mapping to the proprietary Athena database. Users have the option of standard protocols for analysis or advanced protocols that enable additional taxonomic classification capabilities.
The Athena Microbial Reference Database features:
- ~66,000 highly curated 16S-ITS-23S gene regions representing over 20,000 well-annotated microbial genomes
- Continuous updating with new genomic information with the option for users to add their own reference gene sequences
Key Software Features
- Point-and-click, GUI interface that runs on laptop or server
- Option to deploy a CentOS command line
- Automated demultiplexing for all PacBio or Oxford Nanopore compatible kits (16S or 16S-ITS-23S)
- Accurate read mapping to the Athena Database with Excel based and FASTQ file data outputs
- Classification of unknown microbes by scanning for related genomes
- Categorization of the highest species and strain taxonomic annotation using innovative algorithms
- Advanced analysis protocols available for improved error correction and tracking of novel microbes*
*Amplicon sequence variant (ASV) assignment using DADA 2 with StrainID
SBanalyzer Quick Start Guide
The SBanalyzer is set up to be a point and click software, and this video is a quick start guide on how to use the software to analyze results of a sequencing run. Click here to access the guide.
SBanalyzer software* installation on Microsoft Windows 10 or Linux CentOS 7 or 8
1TB disk drive (2 TB recommended)
32 GB RAM (64 for multiple simultaneous Sequel2 run analysis)
Processor Options: Six core i7 4 GHz minimum.
Microsoft Excel for Windows 10, Centos 8 GUI with LibreOffice
Can be run on Oracle VM with Centos 8 installed on Mac or Windows
Server with 32-core head node, 64GB RAM, 2TB hard drive is even better
*Software license required