Shotgun metagenomics is a next-generation sequencing approach used to characterize the composition of complex, multispecies samples. Total RNA or DNA is extracted from a sample, fragmented, and sequenced to gather information on the contents of the sample. While DNA sequencing (metagenomics) has been more common, RNA sequencing (metatranscriptomics) is growing in importance because it better represents metabolically active organisms, provides vital information on active gene pathways, and can be used to understand the effect on gene expression of changing environmental conditions or treatments.
Genes with low expression are often barely detectable because of the overabundance of ribosomal RNA in bacterial and human samples.
With CRISPRclean, more than 99% of human ribosomal RNA and over 200 bacterial species is removed. This improves species detection by eliminating the noise and allowing greater sensitivity for low-expressing genes.
The samples and method
Clinical Stool Samples
Two patient stool samples were spiked with 600 or 60 copies of synthetic SARS-CoV-2 control. NEB Ultra II Directional RNA Library Prep Kits were used to prepare libraries, and then a CRISPRclean Pan-Bacterial rRNA Depletion Kit was applied to the samples to remove ribosomal RNA sequences from human and more than 200 bacterial species. The libraries were sequenced on short-read sequencing instrument. Data analysis was performed using Jumpcode proprietary software to measure alignment and depletion rates, the Silva database for ribosomal RNA read alignment, and the Kraken2 and the CosmosID pipelines for k-mer based metagenomic investigation.
Increased detection of bacterial species by 300-400%.
Removal of ribosomal RNA greatly increased detection of bacterial species in patient stool samples treated with CRISPRclean depletion products.
More unique k-mers identified in samples treated with CRISPRclean.
Samples treated with ribosomal RNA depletion show an increase in unique k-mer frequency.
Increased bacterial composition of CRISPRclean-treated samples compared to untreated samples.
CosmosID output using frequency based show a large increase in detection of bacterial (non-blue) composition of the CRISPRclean-treated sample compared to the untreated sample.