Background: In a subset of renal transplant recipients with urothelial carcinoma, tumor cells show universal expression of BK Polyomavirus (BKPyV) proteins, a hallmark of oncoviruses. We previously validated 26 sets of fully nested primers providing overlapping coverage of the BK genome, using Sanger Sequencing. Next Generation Sequencing (NGS) offers several advantages over Sanger Sequencing, including high-throughput processing, multiplexing, and identification of genomic integration sites using linker-adapters.

Methods: DNA was extracted from archived FFPE blocks of a bladder cancer expressing BKPyV proteins (Qiamp Gene Read FFPE kit, QIAGEN). To generate a library, 500 ng of DNA was sheared to a median length of 571 bp. After end processing with T-overhangs, Tru-Seq barcoded Y-adapters (Illumina) were ligated. To enrich for BKPyV sequences, the library was divided and amplified in 3 sequential PCR reactions using 6 separate pools of tiled primers. The 1^st and 2^nd reactions used nested BKPyV primers and Illumina Primer 2 (30 cycles each). The 3^rd PCR reaction extended a 5' Illumina stub sequence on the internal primer into a full Illumina universal adapter sequence (4 cycles). Primers and single-strand products were removed after each step (DNA Clean and Concentrator, Zymo Research). Paired end sequencing was then performed using a MiSeq Reagent Nano kit v2 on a MiSeq sequencer (Illumina). The output was filtered for quality and read length, adapter and BK primer sequences were trimmed, and the sequence was mapped against the hg19 and BKPyV genomes.

Results: 80% of sequences align with the BKPyV genome. After mapping, the sequence output shows 93.3% coverage of the BKPyV genome. A genomic integration site is not definitively identified, but there is a large sequence gap between 1689 and 1933. The median read depth is 626. Polymorphisms in the viral genome identify it as a clade 1b1 virus, confirmed by Sanger sequencing.

Conclusion: BKPyV specific PCRs were successful in enriching for BKPyV sequences in a human genomic sample obtained from FFPE tissue archived for nearly a decade. The inclusion of a barcode in the adapter has the potential to allow multiple samples (8-16) to be simultaneously analyzed. Primer revision may help resolve coverage gaps and identify integration sites in future experiments. Structural analysis of BKPyV genomes and integration sites may help shed light on the mechanism of oncogenesis of BKPyV in some transplant recipients.

CITATION INFORMATION: Harake E., Wu W., Opp J., Farkash E. Y-Adapter Mediated Next Gen Sequencing of BKPyV from Archived FFPE Tissue Am J Transplant. 2017;17 (suppl 3).

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