Metagenome

Metagenome Sequencing

Next generation sequencing (NGS) has proven to be an invaluable tool for investigating diverse environmental and host-associated microbial communities, helping to generate enormous new data sets that can be mined for information on the composition and functional properties of vastly great numbers of microbial communities.

The applications of NGS in microbial community profiling include amplicon sequencing (typically 16S rRNA sequencing for bacteria and 18S rRNA/ITS sequencing for fungi), metagenomic shotgun sequencing, metatranscriptomic sequencing and viral metagenomics sequencing, which can help to answer the questions of ‘who is present in the community’, ‘what could they be doing’ and ‘how these microorganisms interact’.

16s rRNA / 18s rRNA / ITS / Amplicon Sequencing

16s rRNA gene, a highly conserved component, is the most widely used gene marker for genus and species identification and taxonomic significance in bacteria and archaea. The estimated substitution rate for hypervariable regions is approximately 7000 times higher than the highly conserved ones, which contains abundant taxonomic information based on these genetic differences. Therefore, the 16S gene amplicons obtained from PCR can be used to deduce taxonomic identifications based upon bioinformatics alignments.

Nevertheless, 18S rRNA is commonly used in fungi for phylogenetics since it has more hypervariable domains than 16S. In addition to this, the ITS (Internal Transcribed Spacer) region (including 5.8S), removed in the posttranscriptional process of nuclear rRNA cistron, has been widely regarded as a universal fungi barcode marker for a successful identification for the broadest range of fungi. And compared to 18S, ITS is more variable and hence more suitable as the genetic marker for measuring intraspecific genetic diversity.

16S/18S/ITS Amplicon Sequencing has now been a well-established method for microbial identification and phylogeny studies of samples from complicated microbiomes or environments. In addition to next-generation sequencing platforms, ONEOMICS also provides full-length 16S/18S/ITS amplicon sequencing by using PacBio SMRT or HIFI long read sequencing technology.

Advantages of 16S/18S/ITS Amplicon Sequencing

· The most common housekeeping genetic markers with conserved and variable regions

· Multiple applications: microbial identification, diversity analysis, taxonomy and phylogeny, new species determination, relationship study of microorganism and disease, metagenomics, etc

Bioinformatics Analysis

· Sequencing data processing

· OTU analysis and species annotation

· Alpha & Beta Diversity analysis

Sample requirements

· Samples sources including faeces, natural environments, as well as DNA samples

· DNA amount ≥ 2 μg (concntration ≥ 30 ng/μl ; volume ≥ 30 μl, OD260/280=1.8~2.0)

Shotgun Metagenome Sequencing

Metagenomic shotgun sequencing targets the entirety of the microbial genetic information contained in an environmental sample. The obtained community taxonomic profile can be further associated with the functional profile of known and unknown organism lineages. The complete sequences of protein-coding genes and full operons in the sequenced genomes can offer invaluable functional knowledge about the microbial communities inhabiting practical ecosystems under study. Metagenomic shotgun sequencing may provide genetic information on potentially novel biocatalysts or enzymes, genomic linkages between function and phylogeny for uncultured organisms, evolutionary profiles of community function and composition and much more.

Advantages of Metagenomic Shotgun Sequencing

· Cultivation-independent

· Provide comprehensive information on community biodiversity and function

· A wide range of applications in countless fields including the food and medical industries, environmental and disease studies, as well as biomarker research

Bioinformatics Analysis

· DNA assembly

· Sample complexity analysis

· Functional annotation

· Alpha and beta diversity analysis

· Gene prediction (KEGG, GO, COG)