Transcriptome sequencing coupled with the PathSeq computational analysis pipeline is highly sensitive and specific for detecting presence of novel and known viruses in primary AIDS-related lymphoma samples. (A) The PathSeq pipeline profiles total RNA by random hexamer priming and high-throughput sequencing. Reads that do not map to the human genome are aligned against known viral and bacterial genomes. The remaining unmappable reads are assembled into contigs to detect transcripts originating from novel viruses. (B) PathSeq is sensitive and specific for detecting the EBV in tumor cells. Viral read count fraction (y-axis) is shown for all ARL samples (x-axis). Using a threshold of 0.01%, we were able to uniquely identify all EBV-positive cases, which were confirmed by in situ hybridization for the EBV encoded RNA 1 (EBER1) transcript. A complete listing of read counts, viral ISH detection, and case identifiers can be found in Table 1. (C) Samples with <0.01% of reads aligning to EBV were profiled by ISH for EBER1 to determine if any of these cases were mislabeled as EBV negative by PathSeq. While scattered EBV⁺ cells infiltrated the tumor, tumor cells were in fact negative for EBV. This is exemplified by cases LY09, LY20, and LY24 with morphologically nontransformed tumor infiltrating lymphocytes highlighted. LY18 is shown to demonstrate EBER1 staining of EBV⁺ samples with EBV RNA in tumor cells.