# MiSeq上机测序时如何准备＜2nM 的文库

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[MiSeq 文库变性和稀释指南](https://support-docs.illumina.com/IN/dnd-wizard/Content/dnd/landing.htm?ins=miseq)中介绍了关于稀释2 nM和4 nM文库标准均一化方法。该稀释步骤可以确保最后使用HT1稀释后NaOH的浓度不高于1 mM。NaOH浓度过高可抑制文库与流动槽（Flow Cell）的杂交，导致簇密度降低。

在文库浓度低于2 nM且高于0.5 nM的情况下也可以进行MiSeq的测序。可通过以下的标准均一化流程[NextSeq 500和550测序系统的文库变性和稀释指南](https://support-docs.illumina.com/IN/dnd-wizard/Content/dnd/landing.htm?ins=nextseq550)将文库稀释到20 pM。这个方案中，可添加200 mM Tris-HCl来中和过量的NaOH，以确保NaOH在最终的溶液中被完全中和。

为了稀释 < 2 nM文库，可按照以下Nextseq 500和Nextseq 550测序系统变性和稀释文库指南中介绍的1 nM或0.5 nM起始文库浓度的步骤，详见图1。当文库稀释到20 pM，按照MiSeq系统文库变性和稀释指南中介绍的稀释变性到20 pM文库后的步骤进行进一步稀释，详见图2。

有关具体仪器的详细说明，参考具体的文库变性和稀释指南。

![](https://supportassets.illumina.com/content/dam/illumina-support/images/bulletins/2021/09/How_to_prepare_2_nM_libraries_for_MiSeq_sequencing_1.jpg)

**图 1.** [NextSeq 500和550测序系统文库变性和稀释指南](https://support-docs.illumina.com/IN/NextSeq550_DnD/Content/NextSeq550/DnD-NS550.htm?protocol=standard)标准均一化方法的变性和稀释步骤

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![](https://supportassets.illumina.com/content/dam/illumina-support/images/bulletins/2021/09/How_to_prepare_2_nM_libraries_for_MiSeq_sequencing_2.jpg)

**图 2.** [MiSeq系统变性和稀释文库指南](https://support-docs.illumina.com/IN/MiSeq_DnD/Content/MiSeq/DnD-MiSeq.htm?protocol=standard)标准均一化方法中对变性后的20 pM文库进一步稀释的步骤

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