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Nature Commus: PippinHT助力UMI-ATAC-seq  技术,提高开放染色质的检测灵敏度


ATAC-seq测序技术简介
通过转座酶获取开放染色质信息的测序技术,英文全称为Assay for Transposase Accessible Chromatin using sequencing。ATAC-seq测序技术的常规流程大致是,细胞或组织样本在核质分离后,将细胞核单独收集在一起,然后通过转座酶对核内的染色质进行打断(转座酶可以将自身结合的一段序列随机插入到基因组中)。紧密包裹的染色质DNA不会被转座酶打断,而开放区域的染色质DNA会被转座酶随机插入并打断。将这些打断后的DNA收集在一起进行后续的建库、测序和分析,即可得到开放染色质的信息(见Fig.1)。开放性染色质是指某个时间点同时进行转录的DNA区域,也被称为可及性染色质。
 
Fig.1  ATAC-seq技术示意图
 
传统ATAC-Seq测序技术的不足之处在于,由于存在PCR扩增步骤,使用标准的ATAC-seq流程无法从PCR扩增产生的PCR duplicates(NGS中由于PCR bias而产生的重复reads被称作duplicates,它是正常测序过程中伴随的一种冗余的误测序产物,由于这些重复序列并不能带来额外信息,相反会影响变异检测结果的准确性,因此下游生信分析中需要去除这些重复序列)中准确地识别出独立产生的相同Tn5插入。
 
改良的UMI-ATAC-seq测序技术

2020年11月份发表在Nature子刊《Communications Biology》上的一篇文章中,来自中国华中农业大学的科研工作者提出对标准的ATAC-seq技术进行改良,将带有标准TruSeq测序接头的独特分子标签(Unique Molecular Identifiers ,简称UMI)添加至标准的ATAC-seq流程中,即UMI-ATAC-seq测序技术(见Fig.2)。 具有相同UMI标签的完全相同的DNA片段是由同一来源模板经过PCR扩增产生的,而带有不同UMI标签的完全相同的DNA片段则可能来自不同的细胞或基因组。因此该技术通过引入独特的分子标签UMIs,能够从PCR duplicates中准确地识别出独立产生(不同细胞/基因组来源的)的但是序列一致的 Tn5插入reads。他们随后证明了UMI-ATAC-seq技术能够更加准确地量化染色质的可及性/开放性,并提高了转录本的识别灵敏度。该文章指出,UMI-ATAC-seq对比标准的ATAC-seq技术,可以保留并识别出约20%被误判为PCR duplicates的有用 reads,避免了有效信息的丢失。其中,文库制备过程中的PCR扩增产物,通过PippinHT实现180-600bp的片段选择,继而使用TruSeq测序引物,在illumina测序平台进行测序分析。
Fig2:The workflow of UMI-ATAC-seq
 
 
 
Sage Science是全球领先的研发制造Pippin系列全自动核酸电泳片段回收仪的生产厂家,拥有美国技术专利,各个型号产品可以高效完成不同长度范围的DNA片段的精准回收,广泛用于二代测序、三代测序以及多种分子生物学相关领域。
 
环亚生物科技有限公司作为美国Sage Science公司在中国区的独家代理商,自2011年以来将Sage Science的产品线引入国内,一直为国内用户提供专业的全自动核酸片段回收系统的安装测试、应用培训,技术支持与售后维护工作,赢得客户的一致好评与信任。环亚生物科技有限公司将一如既往的支持越来越多的Sage Science用户。
 
原文链接:
https://www.nature.com/articles/s42003-020-01403-4

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