INVIEW MICROBIOME PROFILING 2.0
In-depth characterisation of microbial communities of any complexity
INVIEW MICROBIOME PROFILING 2.0 is a one-stop service for the identification of populations of low and high complexity down to the genus level. The product is based on next-generation sequencing (NGS) of multiple hypervariable regions of the 16S rRNA gene or ITS regions. The results are sensitive and specific enough to enable classification of low numbers of rare taxa.
Highlights of INVIEW MICROBIOME PROFILING 2.0
- Use of validated primers from high-impact publications
- Extensive coverage of many 16S rRNA hypervariable gene regions, as well as ITS regions
- Multiplexed analysis of any microbiome
INVIEW MICROBIOME PROFILING 2.0 offers all of the following in one package:
- Automated processing of up to 92 samples per run in parallel
- Amplification of hypervariable or ITS regions by PCR
- Use of validated primers (V1-V3, V3-V5, ITS1 and ITS2)
- Purification of PCR products with reduced chimera formation
- Integration of tagged adaptors by fusion primer PCR
- Preparative gel purification
- Sequencing using Illumina technology
- Results summarised in comprehensive Data Analysis Report
- Available under ISO17025-accredited service standard
Accepted starting material for INVIEW MICROBIOME PROFILING 2.0
- >10 ng DNA with at least 103 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 platform for INVIEW MICROBIOME PROFILING 2.0:
Sequencing Mode for INVIEW MICROBIOME PROFILING 2.0
- 2 x 300 bp paired-end sequencing mode
Bioinformatic analysis included in INVIEW MICROBIOME PROFILING 2.0
- Quality filtering of reads
- Merging of overlapping read pairs
- Redundancy removal (clustering and removal of singletons)
- Chimera check and removal
- RDP-based OTU assignment (BLAST)
Deliverables of INVIEW MICROBIOME PROFILING 2.0:
- Sequences of unique and non-chimeric clusters (.fasta)
- Sequences of discarded clusters / singletons (.fasta)
- Sequences of cluster w/ and w/o OTU assignment (.fasta)
- Read statistics (.tsv)
- OTU tables (.tsv)
- 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 genus abundance
- Demo data available
- 25 days for up to 46 libraries
- 35 days for 47-92 libraries
INVIEW MICROBIOME PROFILING 2.0 enables analysis of genomic regions such as the bacterial 16S rRNA gene (Fig.1) or the fungal internal transcribed spacer (ITS) (Fig. 2). Amplification and high-throughput sequencing of the hypervariable regions of the 16S rRNA gene are particularly suitable for determining the phylogeny and taxonomy of unculturable microorganisms from diverse samples.
Figure 1: Schematic overview of the 16S rRNA gene
Shown here is the sequence identity of the 16S rRNA gene of more than 6,000 bacteria compared to consensus sequence. Dips indicate hypervariable regions. Hypervariable regions (V1-V9) are shown in grey and the conserved regions in orange.
Primer pair 1: covering variable region V1-V3 (approx. 500 bases)
Primer pair 2: covering V3-V5 (approx. 600 bases)
Primer pair 3: covering V1-V8 (approx. 1400 bases)
ITS analysis: Characterisation of fungal microbiomes using internal transcribed spacer regions (ITS)
ITS regions are mainly used to characterise fungi. They vary in sequence and length and are therefore ideally suited for characterising samples such as mycobiomes in environmental and clinical contexts.
Figure 2: Structure of the ITS regions and the positions of the primers (P1 to P4)
The two ITS regions (ITS1 and ITS2) are between the 18S and 28S rRNA genes and are separated by the 5.8S rRNA gene. In eukaryotic genomes the ITS regions vary greatly in size and sequence.
Primer pair 1: covering the ITS1 region (approx. 300-500 bp)
Primer pair 2: covering the ITS2 region (approx. 350-600 bp)
With INVIEW MICROBIOME PROFILING 2.0, long amplicons of up to 570 bp can be achieved by overlapping read pairs (Fig. 3). Large data output can be delivered with rapid turnaround times.
Figure 3: Analysis of long amplicons possible by merging overlapping read pairs
Microbiome Profiling 2.0 can be applied for various industrial, agricultural, medical and environmental science applications:
- Investigating the ecological, medical or industrial relevance of microbiomes
- Analysing phylogeny and taxonomy, particularly of diverse microbiomes
- Exploring complex samples or unculturable organisms
- Finding contaminants or pathogens
GATC Biotech has tested and demonstrated the utility of this approach for the investigation of complex and non-complex food/industrial, environmental and clinical samples. The targeted sequencing of hypervariable 16S rRNA regions allows us to detect bacteria present at an extremely low frequency. For specific, in-depth analyses of fungal samples, we also offer amplification and sequencing for ITS regions. Please read our white papers for more information on sample projects. Please note: The white papers for the environmental and clinical samples as well as the white paper on food quality assurance are based on GS FLX data from Roche. Nevertheless, they are still valid and demonstrate the scientific questions that can be addressed using INVIEW MICROBIOME.
1. What kind of samples can be used?
In general, all types of (DNA) samples can be used for microbiome analysis, such as:
Environmental samples from various sources
Samples of human origin (swabs, faeces, lavage)
Food samples for quality control on microorganisms and pathogen detection in an industrial setting (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 genera present in the analysed sample and the corresponding read counts. In addition, you will receive all of the raw sequencing data.
Demo data of samples from different sources are available here:
Operational Taxonomic Units (OTUs), which are classified by BLAST analysis against a curated RDP database, are represented in table form including taxonomic identification down to the genus level. Only the best hits are considered for OTU assignment.
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.
Validated primers from high impact publications (The Human Microbiome Project Consortium 2012) are used to generate amplicons covering the variable regions V1-V3 and V3-V5.
Our customers prefer the V1-V3 primer pair to the V3-V5 primer pair.
5. What is the necessary coverage for microbiome analysis?
The required sequencing depth for microbiome analysis mainly depends on the complexity of the sample (the number and representation of individual species) and the aim of the project. Low coverage is sufficient if you expect your sample to contain only few different bacteria; higher coverage is needed if you anticipate many different bacteria. When in doubt, we recommend determining the required depth of sequencing by performing a trial on a sub-set of samples.
6. Which organisms can be detected?
Phylogenic characterisation and analysis of microbial communities can be performed for various sample types and organisms. We have tested and demonstrated the utility of this approach for the identification of complex and non-complex food/industrial, environmental and medical samples. Targeted sequencing of hypervariable regions allows us to detect bacteria present at an extremely low frequency. For in-depth analyses of fungal samples, we also offer amplification and sequencing for ITS regions.
7. How do I choose between INVIEW MICROBIOME Profiling 2.0 with Illumina and INVIEW MICROBIOME High Specificity with Pacific Biosciences?
Both 16S rRNA sequencing 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 a short turnaround time.
INVIEW Microbiome High Specificity with PacBio is the method of choice for sequencing 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. What happened to INVIEW MICROBIOME Profiling?
GATC Biotech has finished replacing Roche’s GS FLX, as Roche has discontinued its 454 sequencing business. Analysing ITS and 16S rRNA gene regions on Illumina’s technology guarantees a microbiome analysis that will remain reliable going forward. Our INVIEW Microbiome Profiling 2.0 ensures long-lasting consistency and highly standardised analysis pipelines to guarantee reliable and consistently high-quality results at any point in your project. INVIEW Microbiome High Specificity now makes in-depth analysis available for the first time.
9. 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!