Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing.
Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing.
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DOI：
10.1261/rna.078703.121
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发表时间：
2022-03
期刊：
影响因子：
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通讯作者：
Ettwiller L
中科院分区：
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作者：
Mulroney L;Wulf MG;Schildkraut I;Tzertzinis G;Buswell J;Jain M;Olsen H;Diekhans M;Corrêa IR Jr;Akeson M;Ettwiller L
纳米孔测序设备直接读取单个RNA链。这有助于识别外显子连接和核苷酸修饰；然而，使用传统的直接RNA纳米孔测序，聚(A) RNA的5 ‘和3 ’端不能明确地识别。这部分是由于体内和体外的RNA降解可以模糊转录起始和结束位点。在这项研究中，我们的目标是在约400万个纳米孔poly(A)选择的RNA reads中鉴定单个全长人类RNA同种异构体。首先，为了鉴定携带5 ' m7G帽的RNA链，我们将生物帽替换为附着在45-nt寡聚物上的修饰帽。这种低聚物自适应方法改进了5 ‘端测序，保证了5 ’ m7G端帽的正确鉴定。其次，在这些5 ‘的纳米孔中，我们筛选了与3 ’聚腺苷化位点一致的特征。结合这两个步骤，我们从人GM12878细胞中鉴定出294,107个高置信度的全长RNA支架，其中大部分（257,721个）与蛋白质编码基因一致。其中，4876个支架显示了未注释的异构体，这些异构体通常位于先前鉴定的较长RNA异构体的内部。m7G帽和开放染色质的正交数据，如CAGE和DNase-HS序列，证实了这些高置信度RNA支架的有效性。
Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional direct RNA nanopore sequencing, the 5′ and 3′ ends of poly(A) RNA cannot be identified unambiguously. This is due in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoforms among ∼4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5′ m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nt oligomer. This oligomer adaptation method improved 5′ end sequencing and ensured correct identification of the 5′ m7G capped ends. Second, among these 5′-capped nanopore reads, we screened for features consistent with a 3′ polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds from human GM12878 cells, most of which (257,721) aligned to protein-coding genes. Of these, 4876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data for m7G caps and open chromatin, such as CAGE and DNase-HS seq, confirmed the validity of these high-confidence RNA scaffolds.