# 为什么Read1测序读长推荐至少需要26个循环？

 

以下指导内容能够为客户测序定制文库时，最大化数据产量和质量。所有Illumina文库设计的Read1测序读长都至少需要26个循环。Illumina也建议设计定制文库时至少需要测序26个循环，以保证可靠的碱基读取与质量打分。

 

如果测序时有设置特异程序，Illumina测序系统是可以进行少于25循环的Read1的测序。但是，我们不推荐也不支持这种设置。Illumina初级分析软件RTA经过优化并且验证，利用Read1前25循环进行质量参数计算。**Illumina 建议对 Read 1 运行至少 26 个循环，以确保所有 RTA 计算完成。**对于Read1少于25个循环的测序，RTA会利用Read1可用循环数计算并报告簇过滤比例与质量分数。但是这些条件下RTA的表现并没有被验证过，这些测序的簇过滤选择条件与报告的质量打分参数与read1读长为25个循环或者更多循环的测序不等同也无法直接进行比较。对于客户read1少于25循环的测序，质量参数需要单独确认。

 

如果定制文库含有碱基低多样性区域，例如内部标签，引物退火位点以及聚合物等，需要考虑加入平衡文库，例如PhiX，以增加碱基多样性，而不是将Read1的测序循环数少于25个循环。定制文库中加入的PhiX比例取决于不同的仪器和文库，可能需要进行优化。当测序文库使用定制测序引物时，如果需要加入PhiX，可以将定制引物掺入到Illumina引物中。下面这篇支持文档提供更多详细信息：[将定制引物掺入到Illumina测序引物中](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000007451)。

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