PDF(Pubmed)
Abstract：
UNASSIGNED: HBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms.
UNASSIGNED: DNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline.
UNASSIGNED: HBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations.
UNASSIGNED: Targeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC.
UNASSIGNED: Fresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.
UNASSIGNED: DNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline.
UNASSIGNED: HBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations.
UNASSIGNED: Targeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC.
UNASSIGNED: Fresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.
摘要：
未经证实：HBV感染全球超过2.57亿人，并与肝细胞癌（HCC）的发展有关。HBVDNA整合到宿主基因组可能是肝癌发生的关键驱动因素。这里,我们利用靶向长读测序来确定HBVDNA整合的结构以及HBVmRNA的完整同工型信息，比传统的下一代测序平台更准确的定量。
未经证实：从GS-US-174-0149临床试验中收集的新鲜冷冻肝活检中分离DNA和RNA。开发了生物素化寡核苷酸的泛基因型面板，以从剪切的基因组DNA（〜7kb）和来自聚腺苷酸化RNA的全长cDNA文库中富集HBV序列。在PacBio长读平台上对样品进行测序，并使用定制的生物信息学管道进行分析。
UNASSIGNED：HBV靶向长读DNA测序产生了跨越整个整合的高覆盖率数据。引人注目的是，在42个样本中的13个（31％），我们能够检测到由2个不同染色体侧翼的HBV序列，表明与HBV整合相关的染色体易位。染色体易位对每个活检样本都是独特的，表明每个都是随机起源的，在某些情况下有克隆扩张的证据。使用靶向长读RNA测序，我们确定，在HBeAg阳性的患者中，超过95％的所有HBV转录本来自cccDNA。相比之下,HBeAg阴性的患者大多从整合中表达HBsAg。
UNASSIGNED：靶向lso-Seq允许HBV转录组的准确定量和转录分配到cccDNA或整合起源。在非HCCCHB患者肝活检中存在多个独特的HBV相关染色体间易位，这表明具有诱变潜力的新机制可能有助于进展为HCC。
UNASSIGNED：对HBV感染患者的新鲜冷冻肝活检进行靶向长读RNA和DNA测序。长读RNA测序捕获整个HBV转录本在一个单一的阅读，允许从HBV基因组重叠转录本的分辨率。该决议使我们能够量化来自整合的转录负担与cccDNA起源于个体患者。HBeAg阳性的患者与HBeAg阴性的患者相比，来自cccDNA的HBV转录组的比例明显更大。长读DNA测序捕获整个整合的HBV序列，包括单个读段内的数千碱基的侧翼宿主序列。该决议使我们能够描述两个不同宿主染色体两侧的整合事件，表明整合的HBVDNA与染色体间易位有关。这可能导致显著的转录失调和驱动进展为HCC。
未经证实：从GS-US-174-0149临床试验中收集的新鲜冷冻肝活检中分离DNA和RNA。开发了生物素化寡核苷酸的泛基因型面板，以从剪切的基因组DNA（〜7kb）和来自聚腺苷酸化RNA的全长cDNA文库中富集HBV序列。在PacBio长读平台上对样品进行测序，并使用定制的生物信息学管道进行分析。
UNASSIGNED：HBV靶向长读DNA测序产生了跨越整个整合的高覆盖率数据。引人注目的是，在42个样本中的13个（31％），我们能够检测到由2个不同染色体侧翼的HBV序列，表明与HBV整合相关的染色体易位。染色体易位对每个活检样本都是独特的，表明每个都是随机起源的，在某些情况下有克隆扩张的证据。使用靶向长读RNA测序，我们确定，在HBeAg阳性的患者中，超过95％的所有HBV转录本来自cccDNA。相比之下,HBeAg阴性的患者大多从整合中表达HBsAg。
UNASSIGNED：靶向lso-Seq允许HBV转录组的准确定量和转录分配到cccDNA或整合起源。在非HCCCHB患者肝活检中存在多个独特的HBV相关染色体间易位，这表明具有诱变潜力的新机制可能有助于进展为HCC。
UNASSIGNED：对HBV感染患者的新鲜冷冻肝活检进行靶向长读RNA和DNA测序。长读RNA测序捕获整个HBV转录本在一个单一的阅读，允许从HBV基因组重叠转录本的分辨率。该决议使我们能够量化来自整合的转录负担与cccDNA起源于个体患者。HBeAg阳性的患者与HBeAg阴性的患者相比，来自cccDNA的HBV转录组的比例明显更大。长读DNA测序捕获整个整合的HBV序列，包括单个读段内的数千碱基的侧翼宿主序列。该决议使我们能够描述两个不同宿主染色体两侧的整合事件，表明整合的HBVDNA与染色体间易位有关。这可能导致显著的转录失调和驱动进展为HCC。
