The discovery of circulating cell-free DNA (exDNA) and RNA (exRNA) in human body fluids, including serum, has sparked great interest in using these nucleic acids as markers of disease, as diagnostic tools or as therapeutic molecules. Here, we examined the exRNA/exDNA profiles in community-dwelling individuals. Surprisingly, we found a considerable percentage of sequenced extracellular DNA/RNA remains unaligned to the human genome/transcriptome compared to DNA/RNA extracted from cells. In this project, we have tried to identify the nature of the unaligned reads after filtering out the sequencing adapters and primers. Total RNA was extracted from the serum of 10+ healthy young individuals and 10+ healthy old individuals and total RNA-seq on the Ion Torrent Proton sequencer was performed using a modified protocol to obtain both small and large sized RNAs. The reads were first aligned to the human genome (hg19) using a two-step process with Tophat2 and Bowie2 (not presented here) and unaligned reads were aligned to all known organisms in the Refseq v70 database (54,118 organisms) encompassing prokaryotes (archaea and bacteria) and eukaryotes (fungi, plants and vertebrate & invertebrate animals). Kraken software was used to align against 618.8 billion DNA sequences and 24.2 billion RNA sequences in a sequential manner of the different kingdoms of life. The results surprisingly show that different individuals have different percentages of fragments of DNA and/or RNA from numerous organisms. Some of the organisms identified are bacteria (including Clostridium sp. and Pseudomonas sp.), fungi (including Malassezia sp., Saccharomyces), plants (such as corn, tobacco, poplar etc.), insects (including mites, deer tick and body lice), vertebrates (such as turkey and cow). This suggests that the species we are identifying in serum may give important information about diet and behavior among the individuals studied. Many of the sequenced reads from the serum of different individuals align to specific regions of the genome of an organism (e.g. Pseudomonas aeruginosa), indicating that perhaps these fragments (kilobases in size) are particularly more stable, which allows them to withstand degradation in body fluids. Thus, far we have identified these organisms through computational methods, but further experimental validation will be performed.
Acknowledgement: This research was supported by Intramural Research Program of the National Institute on Aging, NIH.