*Purpose: The selective targeting of T cells in transplant immunosuppression medications leads to a high rate of viral infections, with different immunosuppression regimens resulting in variable effects on protective immunity. We aim to use next-generation sequencing (NGS) to characterize the microvirome of renal transplant recipients and examine variance in the context of immunosuppression, rejection, and clinically apparent infection.

*Methods: Twenty-two patients were included with a total of 129 plasma samples collected at baseline and regular intervals after kidney transplant. Cell-free DNA was isolated from plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). The isolated DNA was fragmented and appended with dual-indexed bar codes using the NexteraXT DNA Library Preparation kit (Illumina). Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled at equimolar concentrations, and sequenced on an Illumina NovaSeq 6000 at 150PE to a depth of approximately 30 million reads/sample. Low quality reads were computationally removed, and adapters trimmed. Host and contaminant reads were removed, and remaining reads were assembled into contigs using the metaSPAdes metagenomic assembler. The resulting contigs were aligned against the known virome.

*Results: After removal of human, contaminant, and low-quality reads, an average of approximately 400,000 reads per sample remained, representing only 2% of overall reads. Contig assembly was completed with resulting U50 of 366bp and UL50 of 8306. Contigs were filtered to exclude those shorter than 300bp, and viral hits were plotted by relative abundance (adjusted for contig and genome length) over time for each patient. The majority of patients demonstrated a notable expansion of anelloviridae immediately postoperatively that gradually waned with immunosuppression reduction during the first year after transplant. Microvirome composition was similar across immunosuppression regimens. In addition to anelloviridae, herpesviridae, and polyomaviridae, samples also had significant levels of retroviridae and poxviridae.

*Conclusions: The microvirome is an underutilized tool to understand protective immunity in the transplant population. This dataset provides a rigorous characterization of the microvirome in healthy kidney transplant recipients. We plan next to apply these same methods to a cohort of kidney transplant recipients with symptoms characteristic of viremia, but no identified underlying etiology. Using the healthy cohort as a benchmark will allow for analysis of viral differential abundance in affected patients.

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