Metagenome analysis of plasma derived CFDNA in healthy patients and colon diseases

Introduction: There are many theory of the cell free DNA (cfDNA) origin such as apoptosis, necrosis and active secretion; however the exact nucleotide composition is unknown. We are constantly exposed to foreign DNA from various sources like benign or malicious microbes in and on our body, pollens in the inhaled air and as the largest amount with the daily food supply. Here we report a new possible mechanism, which can be contribute the quantity and quality of the cfDNA.

Aims: Our aims were to determine the DNA content of cfDNA by NGS sequencing technology and identify differences among four clinical groups and analyze the unmapped read from NGS sequencing data.

Methods: CfDNA was extracted from plasma samples which were collected (TUKEB 2009/037) from 50 – 50 normal, IBD, colorectal adenoma and -cancer patients. Total of 3 – 5ug cfDNA was pooled from each group and three fractions were separated via electrophoresis and DNA fragments were recovered from the gel slices. Intact DNA above 10kb (1st), the fraction is between 200bp to 10kb (2nd) and nucleosomal DNA (3 rd) fractions were separated. Indexed DNA fragment library resequencing was performed on SOLiD IV system. The sequencing yielded 50 nt long reads a total of 86.6 Gbases. Sequence alignments were performed by SHRiMP and Bowtie aligners.

Results: On average above 70% of the reads were mapped to the human reference genome. The non-aligning reads were aligned to genomes of various other organisms. We have identified several different strains of bacteria. Among them Enterobacteriaceae (Escherichia coli, Shigella sonnei) showed the largest presence with genome coverage comparable to the human genome. Since these bacteria are normally present in the human gut the results suggest that the isolation between the gut a circulatory system is not perfect. Beyond bacteria we were able to identify DNA fragments from plant chloroplasts most probably originated from consumed food, especially in IBD samples.

Conclusion: Though the sporadic occurrence of food related miRNA and DNA in blood has already been reported in previous studies, the presence of fragments which can carry complete genes seems to be novel. Our findings can be change fundamentally our general ideas from the digestion processes and macro molecular transport, furthermore may be contributing to understanding cancer development or making blood based diagnostic markers.