Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.
Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B.
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DOI：
10.1016/j.jhepr.2022.100449
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发表时间：
2022-04
期刊：
影响因子：
--
通讯作者：
Feierbach B
中科院分区：
文献类型：
--
作者：
van Buuren N;Ramirez R;Soulette C;Suri V;Han D;May L;Turner S;Parvangada PC;Martin R;Chan HLY;Marcellin P;Buti M;Bui N;Bhardwaj N;Gaggar A;Li L;Mo H;Feierbach B
乙型肝炎病毒感染全球超过2.57亿人，并与肝细胞癌（HCC）的发展有关。HBV DNA整合到宿主基因组中可能是HCC肿瘤发生的关键驱动因素。在这里，我们利用靶向长读测序来确定HBV DNA整合的结构以及HBV mRNA的完整异构体信息，比传统的下一代测序平台更准确地定量。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。开发了一个生物素化寡核苷酸泛基因型面板，用于从剪切的基因组DNA （~ 7 kb）和从多腺苷化RNA中全长cDNA文库中富集HBV序列。样品在PacBio长读平台上测序，并使用定制的生物信息学管道进行分析。针对hbv的长读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%）中，我们能够检测到2条不同染色体的HBV序列，这表明染色体易位与HBV整合相关。每个活检样本的染色体易位都是独一无二的，这表明每个易位都是随机产生的，在某些情况下有克隆扩增的证据。通过靶向长读RNA测序，我们确定hbeag阳性患者中95%以上的HBV转录物来自cccDNA。相比之下，hbeag阴性的患者主要表达来自整合的HBsAg。靶向lso-Seq允许准确定量HBV转录组，并将转录物分配到cccDNA或整合起源。非HCC CHB患者肝脏活检中存在多种独特的hbv相关染色体间易位，这表明一种具有致突变潜力的新机制可能有助于HCC的进展。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测序在单读中捕获整个HBV转录本，允许从HBV基因组中分离重叠转录本。该决议使我们能够量化个体患者中整合与cccDNA起源的转录负担。与hbeag阴性患者相比，hbeag阳性患者的HBV转录组中来自cccDNA的比例明显更高。长读DNA测序捕获了整个整合的HBV序列，包括单个读段内两侧宿主序列的多个千碱基。这种分辨率使我们能够描述由2个不同的宿主染色体两侧的整合事件，表明整合的HBV DNA与染色体间易位有关。这可能导致显著的转录失调并推动HCC的发展。对CHB患者的新鲜冷冻肝活检进行靶向长读RNA和DNA测序。在三分之一的患者中检测到与HBV整合事件相关的染色体间易位。每个活检样本的染色体易位都是独一无二的，这表明每个易位都是随机产生的。在hbeag阳性的患者中，大部分HBV转录组来自cccDNA。
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. 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. 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. 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. 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. Fresh frozen liver biopsies from patients with CHB were subjected to targeted long-read RNA and DNA sequencing. Inter-chromosomal translocations associated with HBV integration events detected in one-third of patients. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly. A larger fraction of the HBV transcriptome originates from cccDNA in patients who are HBeAg-positive.
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DOI：
10.1093/bioinformatics/btq033
发表时间：
2010-03-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Quinlan AR;Hall IM
通讯作者：
Hall IM
影响因子：
24.5
作者：
Péneau C;Imbeaud S;La Bella T;Hirsch TZ;Caruso S;Calderaro J;Paradis V;Blanc JF;Letouzé E;Nault JC;Amaddeo G;Zucman-Rossi J
通讯作者：
Zucman-Rossi J
影响因子：
25.7
作者：
Tong S;Revill P
通讯作者：
Revill P
影响因子：
14.9
作者：
Frankish A;Diekhans M;Ferreira AM;Johnson R;Jungreis I;Loveland J;Mudge JM;Sisu C;Wright J;Armstrong J;Barnes I;Berry A;Bignell A;Carbonell Sala S;Chrast J;Cunningham F;Di Domenico T;Donaldson S;Fiddes IT;García Girón C;Gonzalez JM;Grego T;Hardy M;Hourlier T;Hunt T;Izuogu OG;Lagarde J;Martin FJ;Martínez L;Mohanan S;Muir P;Navarro FCP;Parker A;Pei B;Pozo F;Ruffier M;Schmitt BM;Stapleton E;Suner MM;Sycheva I;Uszczynska-Ratajczak B;Xu J;Yates A;Zerbino D;Zhang Y;Aken B;Choudhary JS;Gerstein M;Guigó R;Hubbard TJP;Kellis M;Paten B;Reymond A;Tress ML;Flicek P
通讯作者：
Flicek P
影响因子：
50.3
作者：
Lau, Chi-Chiu;Sun, Tingting;Wong, Nathalie
通讯作者：
Wong, Nathalie