## 2 Results
### RNA Sequencing Analysis of 5' Capped RNA Identifies Upresulated Unannotated Pol II Transcripts  
Using the open source software program Cufflinks we identified more than 1500 unannotated transcriptionally active regions in HCV infected Huh 7.5 cells that produced 5' capped RNA (data not shown) [31]. These regions were a minimum of 2 kilobases from well annotated genes in the Ensembl 62 (Hg19) database. Some of these transcripts may represent alternative 5' start sites or alternative 3' poly(A) sites of annotated genes. We further refined this list of unannotated transcripts to focus on RNAs that were differentially expressed in HCV infected compared to control cells after 48 and 72 hours of infection with an FDR of \(\geq\)13 and a fold change of 1.5. We also used an RPKM (reads per kilobase of gene exon length per million reads) cutoff of 5 to identify novel RNAs that were moderately to highly expressed (Table 4). One of the unannotated Pol II transcripts was upregulated by 5.0-fold at 48 and 7.5-fold at 72 hours after HCV infection and originated from a 39 kilobase region on chromosome 8 (Figure 8, panel A). qPCR analysis was performed on six additional replicates of HCV and mock infected cell cultures and showed a 2.5-fold increase in this RNA (_p_-value \(<\) 0.05) at the 72 hours (Figure 8, panel B). \begin{table}
\begin{tabular}{|c|c|c|c|c|c|c|c|} \hline \multicolumn{6}{|c|}{**GENOME POSITION**} \\ \hline \multirow{2}{*}{**Chr**} & \multirow{2}{*}{**Start**} & \multirow{2}{*}{**Stop**} & **Total** & **HCV** & **MOCK** & **Fold** & \multirow{2}{*}{**FDR**} & **Time** \\  & & **BPs** & **RPKM** & **RPKM** & **Change** & & **point** \\ \hline
**8** & 47528429 & 47529270 & 841 & 7.74 & 0.93 & 7.52 & 113.0 & 72 h \\ \hline
**5** & 85865845 & 85866626 & 781 & 10.08 & 3.37 & 2.92 & 57.2 & 72 h \\ \hline
**4** & 1384494 & 1385183 & 689 & 6.10 & 2.27 & 2.59 & 21.4 & 72 h \\ \hline
**8** & 87354779 & 87355196 & 417 & 7.95 & 3.04 & 2.50 & 14.7 & 72 h \\ \hline
**16** & 31580193 & 31580636 & 443 & 10.55 & 4.19 & 2.44 & 21.4 & 72 h \\ \hline
**17** & 7967452 & 7968557 & 1105 & 6.15 & 2.62 & 2.33 & 101.1 & 48 h \\ \hline
**16** & 21513065 & 21513758 & 693 & 8.71 & 3.94 & 2.17 & 21.9 & 72 h \\ \hline
**15** & 64995200 & 64995463 & 263 & 31.29 & 15.07 & 2.07 & 95.1 & 48 h \\ \hline
**11** & 122489643 & 122489913 & 270 & 9.48 & 4.76 & 1.97 & 18.1 & 48 h \\ \hline
**14** & 65747331 & 65747885 & 554 & 11.64 & 6.17 & 1.86 & 15.4 & 72 h \\ \hline \end{tabular}
\end{table} 
Table 4: Unannotated transcripts increased during acute HCV infection. It displays ten unannotated transcripts with their respective chromosome location, transcript size, fold change and FDR. RPKM = reads per kilobase per million reads. \begin{table}
\begin{tabular}{|l|c|c|c|c|c|c|} \hline \multirow{2}{*}{**KEGG Pathway name**} & \multirow{2}{*}{**Genes in Pathway**} & \multicolumn{2}{c|}{**6 h**} & \multicolumn{2}{c|}{**48 h**} & \multicolumn{2}{c|}{**72 h**} \\ \cline{3-8}  & & **DEGs** & \(p\) **value** & **DEGs** & \(p\) **value** & **DEGs** & \(p\) **value** \\ \hline
**Ubiquinone and other terpenoid-quinone biosynthesis** & 7 & 1 (5) & 0.03 & & & & \\ \hline
**Taurine and hypotaurine metabolism** & 10 & 1 (5) & 0.03 & & & \\ \hline
**Neurotophin signaling** & 127 & & & & 18 (94) & 0.006 \\ \hline
**One carbon pool by folate** & 18 & & & & 5 (17) & 0.03 \\ \hline
**Prion diseases** & 36 & 2 (21) & 0.006 & & & \\ \hline \end{tabular}
\end{table} 
Table 3: _Cont._Further bioinformatic analysis of this chromosome 8 region revealed that it has sequence homology to the recently identified asparagine synthetase pseudogene 1 (_ASNSP1_). We performed 5' and 3' rapid amplification of cDNA ends (RACE) to further characterize this Pol II RNA. The analysis showed that the Pol II RNA originating from chromosome 8 contains multiple splice variants with the largest variant extending beyond the boundaries of the recently identified pseudogene. Using the ORF Finder software (Softberry) we found a small peptide of 48 amino acids encoded in this unannotated transcript [33].