Easy Analysis for Complex Samples

The microbiome is a diverse system of microbiota living on our skin, in our gut, in our blood, and in the environment. These bacteria, fungi and viruses live in harmony with one another and with us and have a major impact on human health. Microbiome research varies between how the microbiota affects disease development and progression, impacts growth and development, and even mental health including anxiety, depression and autism. Identification of the microflora is an integral part of the study of the microbiome- you must identify what’s there to understand how it’s changing.

Bacterial identification is often done by either shotgun sequencing or 16s sequencing. Shotgun sequencing surveys the entire genome of all the organisms present in a sample. If the goal of your study is to evaluate bacterial diversity of a sample, shotgun sequencing can provide taxonomic information down to the species level and estimate abundance, allowing you to monitor changes. However, it is expensive and requires bioinformatics tools to analyze the data and may result in more data than is necessary for your experiment.

16S rRNA, on the other hand, may not be enough resolution. The 16S rRNA gene is about 1500 bases long and is highly conserved amongst bacteria. However, the 9 hypervariable regions throughout can be used to establish genus and potentially species level identity. Current methods for 16S sequencing focus on 1-2 regions that display genus level variability for sample diversity identification. However, there’s not enough differentiation in these few regions to decipher between similar species. That’s where EZ Species can help.

EZ Species combines the throughput and low cost of 16S sequencing with the resolution of shotgun sequencing. We are still only sequencing the 16S hypervariable regions, but instead of analyzing 1-2, we can review all 8. Multiplex PCR is performed using primers upstream of all 8 hypervariable regions, generating amplicons for sequencing. Once these are sequenced, the reads are aligned to a curated database containing sequences for all 8 hypervariable regions for all species of bacteria. Sequencing results that map to 4+ hypervariable regions are used to generate a list of candidate species per sample. This sub-database is then used to double check the original sequencing results while also serving as a list of discriminatory variable regions for each species. The discriminatory variable region is the region for each species that is most unique to that candidate species. This sequence is used to make the final species identification call.

All of this analysis, filtering and reanalysis occurs in our custom bioinformatics pipeline. In addition to the sample diversity identification, the algorithm also estimates species abundance in each sample allowing you to monitor changes in microflora population due to treatment for example, or genetic variation. As part of the EZ Species package, customers receive sequencing data, taxonomic tables and plots, and an analysis report highlighting the key pieces of data resulting from your experiment – including species level identification for all samples and the relative abundance of each species. EZ Species gets its name from how easy it is to submit your samples and get your data – simply provide gDNA in a 96-well pate, send it in to our lab at Canopy Biosciences and let us do the rest!

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