Kaposi's sarcoma-associated herpesvirus is the etiologic agent of Kaposi's sarcoma and two B-cell malignancies. Recent advancements in sequencing technologies have led to high resolution transcriptomes for several human herpesviruses that densely encode genes on both strands. However, for KSHV progress remained limited due to the overall low percentage of KSHV transcripts, even during lytic replication. To address this challenge, we have developed a target enrichment method to increase the KSHV-specific reads for both short- and long-read sequencing platforms. Furthermore, we combined this approach with the Transcriptome Resolution through Integration of Multi-platform Data (TRIMD) pipeline developed previously to annotate transcript structures. TRIMD first builds a scaffold based on long-read sequencing and validates each transcript feature with supporting evidence from Illumina RNA-Seq and deepCAGE sequencing data. Our stringent innovative approach identified 994 unique KSHV transcripts, thus providing the first high-density KSHV lytic transcriptome. We describe a plethora of novel coding and non-coding KSHV transcript isoforms with alternative untranslated regions, splice junctions and open-reading frames, thus providing deeper insights on gene expression regulation of KSHV. Interestingly, as described for Epstein-Barr virus, we identified transcription start sites that augment long-range transcription and may increase the number of latency-associated genes potentially expressed in KS tumors.

中文翻译：

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卡波西肉瘤相关疱疹病毒转录组的高密度分辨率鉴定了由长程转录和选择性剪接产生的新转录亚型


卡波西肉瘤相关疱疹病毒是卡波西肉瘤和两种 B 细胞恶性肿瘤的病原体。测序技术的最新进展已经产生了几种人类疱疹病毒的高分辨率转录组，这些病毒在两条链上都密集编码基因。然而，由于 KSHV 转录本的总体百分比较低，即使在裂解复制期间，KSHV 的进展仍然有限。为了应对这一挑战，我们开发了一种目标富集方法，以增加短读长和长读长测序平台的 KSHV 特异性读长。此外，我们将这种方法与之前开发的通过多平台数据集成进行转录组解析（TRIMD）管道相结合，以注释转录本结构。 TRIMD 首先构建基于长读长测序的支架，并利用 Illumina RNA-Seq 和 deepCAGE 测序数据的支持证据验证每个转录本特征。我们严格的创新方法鉴定了 994 个独特的 KSHV 转录本，从而提供了第一个高密度 KSHV 裂解转录组。我们描述了大量具有替代非翻译区、剪接点和开放阅读框的新型编码和非编码 KSHV 转录亚型，从而为 KSHV 基因表达调控提供了更深入的见解。有趣的是，正如 Epstein-Barr 病毒所述，我们鉴定了转录起始位点，这些位点可以增强长程转录，并可能增加 KS 肿瘤中潜在表达的潜伏期相关基因的数量。