The human ecosystem
The term “microbiome” is used to describe all the microbes in a community, but also the full collection of genes of all the microbes present.
in health and in sickness
The human microbiota is a complex assemblage of the microbes inhabiting many sites in the human body. We live in symbiosis with our microbes: our bodies provide microbes with resources while the microbes provide functions necessary for our health. Dysbiosis leads to pathogenesis.
High throughput sequencing can help us to understand how microbes influence human disease. By sequencing thousands of organisms in parallel, NGS revolutionized microbiology by allowing concurrent analysis of whole microbial communities and identification of strains that may not be found using other methods. Metagenomics approach gives insight in the way microbes interact in communities, the way they respond to their surroundings, the way they change over time and the way these changes impact human health. In prospectives microbiota compositional measurements can be implemented as a part of standard clinical practice and diagnostics for certain diseases or increased susceptibility to certain disorders.
Marker sequencing is a well-established highly targeted approach for microbe identification. 16S rRNA gene is the most common target used to identify microbes without the need to sequence entire genome.The approach allows flexibility for a wide range of experimental designs and reduces costs, as well as turnaround time.
Our workflow begins with purified genomic DNA. Primers are tailed with sequences to incorporate indexing barcodes, and samples are pooled into a single library for sequencing on Illumina MiSeq sequencing system. We generate paired 300 bp reads whose longer lengths provide high-quality full-length gene sequence, ensuring the most accurate classification. Bioinformatics analysis demultiplexes indexed reads, generates FASTQ files, performs OTU clustering and classification at several taxonomic levels: kingdom, phylum, class, order, family, and genus or species. In addition, rarefaction curves are generated.
SHOTGUN METAGENOMIC SEQUENCING
Shotgun metagenomics allows researchers to comprehensively sample all genes in all organisms present in a given complex sample. The method provides information both about which organisms are present and what metabolic processes are possible in the community.
Since the collection of DNA is largely uncontrolled, it is important to remember that the most abundant organisms in the sample are most highly represented in the resulting sequence data. Thus, large sequencing efforts are required to achieve the high coverage needed to fully resolve the genomes of under-represented community members. However, the random nature of shotgun sequencing ensures that many of these organisms, which would otherwise go unnoticed using traditional culturing techniques, will be represented by at least some small sequence segments.
Our workflow begins with purified genomic DNA. PCR-free library preparation is used to generate libraries. After sequencing and quality control procedures, metagenomic sequences are subject to taxonomic analyses and functional characterization, such as gene prediction and functional annotation, which can be used to characterize the biological functions associated with the community and identify novel genes. The results of these various analyses can be compared to those obtained through analysis of other metagenomes to quantify the similarity between communities, determine how community diversity scales with environmental covariates, and identify biomarkers, i.e. taxa and functions that stratify communities of various types.
RNA-seq for identification of both taxonomic composition and active biochemical functions.
While metagenomics reveals which microbes are present and what potential they have, metatranscriptomics disclose their activity by identifying genes that are expressed in a particular microbial environment.
Unbiased RNA-seq enables annotation and quantification of transcripts giving:
- insights in the gene activity diversity and functional/pathway diversity by detecting how many genes are expressed in a community across all species
- estimates of gene expression abundances - detects the highest expressed genes in a specific environmental condition, thus identifying the most important functionality/pathway required to survive. Note that high sequencing depth is required to detect lower expressed genes
- opportunity for biomarker discovery by performing a differential gene expression analysis, i.e. assessing changes in expression levels between different conditions
For every project we want to make sure that the outcome will meet your expectations
Ribosomal RNA depletion is performed for the efficient use of RNA-seq capacity for all samples. Upon depletion, cDNA library is synthesized, samples are index barcoded and pooled for sequencing. Bioinformatics analysis is directed to determin relative abundance of transcripts/enzymes and pathways for each community. Novel transcripts are assembled and differential expression analysis of different communities is performed in order to identify differentially represented pathways across experimental groups.
For all applications final results are delivered in a comprehensive report in addition to raw data and summary of the methods followed. We provide full support on study design to ensure correct sequencing and bioinformatics strategies are used to meet your project goals. Our expert will consult with you about your specific requirements. Any metadata attached to samples will be used to perform statistical analysis in our default analysis pipelines.