INVIEW MICROBIOME HIGH SPECIFICITY
High throughput microbial classification down to the species level
INVIEW MICROBIOME HIGH SPECIFICITY is a next-generation sequencing service that offers characterisation of microbial communities down to the species level based on full-length 16S rRNA gene profiling. The product combines library preparation, sequencing with PacBio long-read technology and reliable BioIT analysis for better separation of species in microbiomes that are difficult to resolve.
Highlights of INVIEW MICROBIOME HIGH SPECIFICITY
- Excellent taxonomical resolution due to high throughput full-length 16S amplicon sequencing
- Classification of microbial communities down to the species level
INVIEW MICROBIOME HIGH SPECIFICITY is a sample-to-data package that offers:
- Processing of up to 10 samples per run in parallel
- Amplification of whole 16S rDNA by PCR
- Use of validated primers (V1-V8)
- Purification of PCR products
- Barcoding of PCR products, pooling and standard adapter ligation
- Library purification and QC
- Sequencing using PacBio technology
- Expert BioIT analysis for a comprehensive Data Analysis Report
- Available under ISO17025 accreditation
Accepted starting material for INVIEW MICROBIOME HIGH SPECIFICITY
- >10 ng DNA with at least 10e3 bacterial/fungal genomes per sample and PCR amplification
- DNA isolation available as additional service for sand, soil, sludge , stool, urine culture, blood culture, milk, yoghurt (<3.5% fat), buccal swab, skin swab and tracheal fluid
Please note that only S1-classified material is accepted for RNA isolation ordered online. More information about the current rules for classifying biological material can be found here. Please contact us for further information on isolating RNA from material classified as S2.
Sequencing Technology for INVIEW MICROBIOME HIGH SPECIFICITY
- PacBio SMRT Sequencing
Bioinformatics analysis included in INVIEW MICROBIOME HIGH SPECIFICITY
- Quality filtering of sequencing reads
- Removal of chimeras
- Ribosome Database Project-based OTU assignment (BLAST)
- Determination of Shannon and Simpson diversity indices
- Combined OTU analysis
- Filtering and consolidation
- Raw data (fastq, bas.h5, metadata.xml)
- Sorted "Reads of Insert" (fastq)
- Non-chimeric and quality filtered sequences (fasta)
- Sequences w/ and w/o OTU assignment (fasta)
- Read statistics (tsv)
- OTU tables (tsv) and OTU distribution plots (png)
- Shannon diversity index tables (tsv)
- Simpson diversity index tables (tsv)
- Diversity index plots (png)
- Comprehensive Data Analysis Report (pdf)
Sequencing data delivered:
- All raw sequencing data (.fastq)
- Tables listing all genera present in the analysed sample and the corresponding read counts
- Diversity indices
- Data delivery in common file types (.tsv, .txt), which standard software can handle for further analysis or visualisation
- Visualisation of relative species abundance
- 22 days for up to 10 samples per SMRT cell
- Express delivery available: 14 days for up to 10 samples per SMRT cell
INVIEW Microbiome HIGH SPECIFICITY applies an amplicon-based targeted resequencing method based on PacBio’s SMRT sequencing technology. The full-length 16S rRNA gene-sequencing service provides a ready-to-use solution for researchers who are seeking to explore the taxonomy and phylogeny of species from various sources and from samples which are difficult to study using conventional profiling techniques.
PacBio’s technology enables full-length 16S amplicon sequencing. Ultra-long reads in a combination with the new Reads of Insert (ROI) approach results in remarkable read quality. The data generated allow for analysis of microbial communities down to the species level at exceptionally high taxonomic resolution. Pac Bio’s barcoding feature makes it possible to pool up to 10 samples.
Figure 1: Schematic overview of the 16S rRNA gene
Specific applications of INVIEW MICROBIOME HIGH SPECIFICITY include:
- Identification of disease-causative agents and pathogens
- Species-level chacterisation of bacteria that are unculturable or biochemically unidentifiable
- Routine detection of medically important bacteria
- Reference identification for unusual strains
GATC Biotech has tested and demonstrated the utility of this approach for a combined test pool of a number of bacterial species. Targeted sequencing of full-length 16S rRNA regions enables the precise detection of microorganisms at exceptionally high taxonomic resolution. Please read our white paper for more information on this project example.
Are you interested in other gene regions? Please see "Related Products" below.
1. What kind of samples can be used?
A wide variety of (DNA) samples can be used for microbiome analysis, such as:
- Environmental samples isolated from all kinds of sources
- Human samples, such as swabs, faeces, lavage
- Food samples for quality control and pathogen detection in industrial settings like dairies, breweries, meat-processing and agricultural facilities
2. How much starting material is required?
For information on the quantity and concentration required for specific library preparation methods please refer to your quote or contact us. For optimum results, we generally require >10 ng of double-stranded, purified DNA for PCR amplification (the total amount depends on the proportion of microbial DNA in the sample). Moreover, the DNA has to be RNA-free with an OD 260/280 of >1.8 and OD 260/230 of >1.9.
3. What data will I receive?
GATC Biotech will deliver tables (tsv, txt) listing all species present in the analysed sample and the corresponding read counts. In addition, you will receive all of the raw sequencing data.
Operational Taxonomic Units (OTUs), which are classified by BLAST analysis against a curated RDP database, are represented in a table format, including taxonomic classification down to the species level. Only the best hits are considered when assigning OTUs.
A demo data report can be viewed here.
4. Why is the 16S rRNA gene used for bacterial classification?
The 16S rRNA gene is ubiquitous among all bacteria. Its relatively small size of approximately 1,500 bp and its alternating structure of conserved and hypervariable regions (V1-V9) make the 16S rRNA well suited for identification and phylogenetic analysis of microorganisms. Excellent results are achieved with the V1-V8 primer pair. The option of using the V1-V9 primer pair is also available, but discouraged due to unstable primer attachment to the much smaller V9 region.
5. What is the necessary coverage for microbiome analysis?
The actual required sequencing depth mainly depends on the desired sensitivity and the complexity of the sample. The current sequencing coverage is about 15,000 reads per run. When in doubt, we recommend determining the required depth of sequencing by performing a trial on a sub-set of samples. The same applies to the experimental setup for pooling up to 10 samples.
6. Which organisms can be detected?
Phylogenic characterisation and analysis of microbial communities can be performed for various organisms from complex and non-complex food, industrial, environmental and clinical samples. However, not all organisms can be fully resolved using the 16S rRNA gene. The genus Bacillus is one bacterium that cannot be separated through 16S rRNA gene sequencing alone. We can overcome such technical limitations with the use of complementary detection methods with our Customised Solutions service, which offers the same quality as our standardised services.
7. How do I choose between INVIEW MICROBIOME Profiling 2.0 with Illumina and INVIEW MICROBIOME High Specificity with Pacific Biosciences?
Both services benefit from optimised PCR conditions with extremely low chimera formation.
The 2 x 300 bp paired-end read mode makes INVIEW Microbiome Profiling 2.0 ideally suited for sequencing several hypervariable regions at once. Scalable sequencing coverage allows for high sequencing depth as needed. The use of the latest Illumina chemistry provides a high degree of sensitivity and allows for the detection of even very low amounts of taxa in a complex sample. Furthermore, multiplexing makes it possible to process a huge amount of samples in parallel and within in a short turnaround time.
INVIEW Microbiome High Specificity with PacBio is the method of choice for sequencing of the entire 16S rDNA at the highest possible taxonomic resolution. Long amplicon sequencing allows for a high degree of specificity down to the species level. The Reads of Insert approach results in high-quality data output, where the threshold can be defined beforehand. The high degree of sensitivity of the full 16S analysis with SMRT sequencing technology makes it possible to characterise the microbial community, even at low densities.
8. Where should I send my samples?
Please post your samples to:
GATC Biotech AG
European Genome and Diagnostics Centre
Please do not use GATC Collection Points for shipping NextGen samples, because this will delay sample arrival at the appropriate destination!