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转录组重构的不确定性

大多数人类基因经过可变剪接1,因而了解哪种异构体在样本中表达对准确的研究和分析而言至关重要。一个基因可能编码数量多得令人吃惊的蛋白质 – 在某些情况下甚至有着相反的生物学功能2。之前的研究已将剪接突变与各种疾病表型相关联,而许多人类疾病都与可变剪接异构体的水平变化相关联3。然而,利用短读长数据来重构完整异构体的工具缺乏灵敏度和特异性,并使得RNA测序数据的解释变得更复杂4

单分子实时(SMRT)测序可提供获取完整异构体信息所需的长序列,而无需组装或复杂算法。Iso-Seq应用可鉴定转录本,并揭示新的基因异构体,包括基因融合以及全长的mRNA和lncRNA转录本。您可以利用Iso-Seq方法:

  • 直接测序全长转录本,而不需要重构转录本
  • 广泛或靶向研究转录本多样性,以获得关键信息
  • 观察等位基因特异的基因表达
  • 区分不同细胞、组织和疾病状态之间的异构体表达

工作流程:从RNA到全长转录本

Iso-Seq的样本制备

利用 PacBio系统 进行开展SMRT测序

  • 利用Sequel系统 简化样本制备流程和降低项目成本
  • 根据项目需求来扩大或缩小通量
    • 每个SMRT Cell 1M多达20Gb,或250到350 k的全长非嵌合读取序列 *
    • 分析单个SMRT Cell中的多个转录本
  • 探索Sequel系统上1到-2个SMRT Cell中的转录组
  • 提高测序深度,以便更全面地鉴定转录组
利用SMRT AnalysisPacBio DevNet开展数据分析

*读长,读取序列数,每个SMRT Cell产生数据,和其它测序表现结果会根据样品的类型/质量,插入片段长度以及其它因素产生变化

 

特色研究:鉴定去势抵抗性前列腺癌(CRPC)中的新全长异构体

androgen receptor isoform sequencing

科学家利用长读长测序来鉴定和定量在CRPC中表达的全长雄激素受体(AR)异构体。他们鉴定出一个截短的异构体AR-V9,与原发性耐受AR靶向治疗的标志物AR-V7相比,AR-V9是更具潜力的生物标志物。深入了解这项研究。

Clark, T., et al., (April, 2017) SMRT Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants. Poster presented at American Association for Cancer Research. Washington D.C.

若有意了解如何利用Iso-Seq分析转录组的复杂性,请联系我们

References

  1. Pan Q., et al., (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nature Genetics. 40, 1413-1415.
  2. Schwerk, C. and Schulze-Osthoff, K. (2005) Regulation of apoptosis by alternative pre-mRNA splicing. Molecular Cell. 19(1), 1-13.
  3. https://www.eurasnet.info/scientists/alternative-splicing-and-disease/list-of-diseases, accessed on 8/3/2015
  4. Korf, I. (2013) Genomics: the state of the art in RNA-seq analysis. Nature Method. 10(12), 1165-1166.

Selected Resources