Human transcriptome subtraction by using short sequence tags to search for tumor viruses in conjunctival carcinoma

Abstract

Digital transcript subtraction (DTS) was developed to subtract in silico known human sequences from expression library data sets, leaving candidate nonhuman sequences for further analysis. This approach requires precise discrimination between human and nonhuman cDNA sequences. Database comparisons show high likelihood that small viral sequences can be successfully distinguished from human sequences. DTS analysis of 9,026 20-bp tags from an expression library of BCBL-1 cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) resolved all but three candidate sequences. Two of these sequences belonged to KSHV transcripts, and the third belonged to an unannotated human expression sequence tag. Overall, 0.24% of transcripts from this cell line were of viral origin. DTS analysis of 241,122 expression tags from three squamous cell conjunctival carcinomas revealed that only 21 sequences did not align with sequences from human databases. All 21 candidates amplify human transcripts and have secondary evidence for being of human origin. This analysis shows that it is unlikely that distinguishable viral transcripts are present in conjunctival carcinomas at 20 transcripts per million or higher, which is the equivalent of approximately 4 transcripts per cell. DTS is a simple screening method to discover novel viral nucleic acids. It provides, for the first time, quantitative evidence against some classes of viral etiology when no viral transcripts are found, thereby reducing the uncertainty involved in new pathogen discovery.

FIG. 1.

Schematic diagram for in silico subtraction of BCBL-1 L-SAGE tags. L-SAGE was performed on BCBL-1 cell line RNA to generate 9,026 20-bp tags. HiFi sequences from two or more tags were chosen after the exclusion of ambiguous tags and single-reading tags from the total group of tags. HiFi sequences were then compared against human RefSeq database (RNA and genomic) entries downloaded from the NCBI website for exact and near-exact (19-in-20-base) matches. The identities of KSHV T1.1 and K12 sequences were confirmed by 3′ RACE. Hum, human.

FIG. 2.

In silico subtraction analysis of SCCC transcriptome. Three separate L-SAGE libraries were generated from SCCCs (two AIDS associated and one non-AIDS associated), resulting in a combined 241,122-SAGE-tag pool and 18,204 unique HiFi sequences. Sequential subtraction generated 146 sequences that were compared to EST and NR database entries to generate a candidate list of 21 HiFi sequences. These 21 sequences were examined by rSAGE and 3′ RACE for evidence of human origin. Hum, human.

FIG. 3.

Advantages that distinguish DTS as a valuable tool for identifying conditions with viral etiologies are listed, along with factors to consider in interpreting DTS results.