# 通过% Occupied和% Pass Filter散点图优化NovaSeq 6000和X、MiSeq i100和iSeq 100上样浓度 NovaSeq X系列、MiSeq i100、NovaSeq 6000和iSeq 100测序平台使用的是有一定数量纳米孔的patterned flowcell，文库在孔中进行簇生成。正是由于这一特点，当比较同一测序平台的测序结果时，不同批次测序之间的原始簇密度是相同的。为了优化NovaSeq 6000和iSeq 100测序平台的上样浓度，可以通过Sequencing Analysis View (SAV)软件绘制% Occupied和% Pass Filter的散点图来确定测序时上样浓度属于过低、最佳，还是过高。可以按照下面的说明绘制这些指标的散点图并根据散点图结果优化文库上样浓度。 若要查看“% Occupied”数据，NovaSeq 6000需要SAV版本在v2.4.5以上，iSeq 100需要SAV 版本在v2.4.7以上。查看NovaSeq X系列和MiSeq i100系列测序平台数据则需要SAV版本在v3.0以上。SAV安装程序可以从 [Sequencing Analysis Viewer Support](https://support.illumina.com/sequencing/sequencing_software/sequencing_analysis_viewer_sav.html) 页面下载 关于如何创建% Occupied和% Pass Filter散点图的视频教程可以在[这里](https://v.youku.com/v_show/id_XNTgzNTAzNzU0NA==.html)找到。 注: % Occupied这一指标不适用于Illumina其他测序平台的数据。 **利用SAV v2.4.x绘制% Occupied和% Pass Filter的散点图：** 1\. 将运行数据加载到SAV中，并选择**Imaging**选项卡: ![](https://supportassets.illumina.com/content/dam/illumina-support/images/bulletins/03/Plotting%20%%20Occupied%20by%20%%20Pass%20Filter%20to%20optimize%20loading%20concentration%20for%20NovaSeq%20and%20iSeq%20100%20platforms_1.png) 2\. 从工具栏中选择散点图绘图工具，弹出一个绘图窗口。 ![](https://supportassets.illumina.com/content/dam/illumina-support/images/bulletins/03/Plotting%20%%20Occupied%20by%20%%20Pass%20Filter%20to%20optimize%20loading%20concentration%20for%20NovaSeq%20and%20iSeq%20100%20platforms_2.png) 3\. 在窗口右侧的Data选项卡中，“X Values”选择“**% Occupied**”，“Y Values”选择“**% Pass Filter**”。散点图将自动生成。 ![](https://supportassets.illumina.com/content/dam/illumina-support/images/bulletins/03/Plotting%20%%20Occupied%20by%20%%20Pass%20Filter%20to%20optimize%20loading%20concentration%20for%20NovaSeq%20and%20iSeq%20100%20platforms_3.png) **利用SAV v3.0绘制% Occupied和% Pass Filter的散点图：** 1\. 将运行数据加载到SAV中，并选择**Imaging**选项卡: ![image.png](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-483cf9d0904daaaeeafd4beff3e5a0e45c68028b%2Fimage1.png?alt=media) 2\. 选择Imaging选项卡下选择**Custom Plot**。 ![image.png](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-f4bbf562dfe23e9b024576967a1f74c69a019439%2Fimage2.png?alt=media) 3\. 在Custom Plot窗口中，确认选中**Scatter Plot**。 4\. 在X Axis Value下拉菜单，选择**% Occupied**。 5\. 在Y Axis Values下拉菜单，选择**% Pass Filter**。 6\. 可选：在**Color By**下拉菜单，可根据各种标准对散点图点进行颜色编码。 ![image.png](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-5e986964daecda721f1acd02d2636f494672ad93%2Fimage3.png?alt=media) **评估散点图：** 上样浓度过低: * 从图的左下到右上，点落在一条斜率为正的直线上，% Occupied和% Pass Filter的变化幅度较大 ![](https://supportassets.illumina.com/content/dam/illumina-support/images/bulletins/03/Plotting%20%%20Occupied%20by%20%%20Pass%20Filter%20to%20optimize%20loading%20concentration%20for%20NovaSeq%20and%20iSeq%20100%20platforms_4.png) 最佳上样浓度: * 在散点图中，点聚集为一堆具有正斜率的点，% Occupied和% Pass Filter的变化幅度较小 ![](https://supportassets.illumina.com/content/dam/illumina-support/images/bulletins/03/Plotting%20%%20Occupied%20by%20%%20Pass%20Filter%20to%20optimize%20loading%20concentration%20for%20NovaSeq%20and%20iSeq%20100%20platforms_5.png) 上样浓度过高: * 点有一些轻微的负斜率、接近垂直，并且% Occupied高达90以上， % Pass Filter的变化幅度较大 ![](https://supportassets.illumina.com/content/dam/illumina-support/images/bulletins/03/Plotting%20%%20Occupied%20by%20%%20Pass%20Filter%20to%20optimize%20loading%20concentration%20for%20NovaSeq%20and%20iSeq%20100%20platforms_6.png) 如果散点图显示上样浓度过低或者过高，则需要分别提高或降低上样浓度，以达到最佳上样浓度。如果散点图显示的是最佳上样浓度，那么在未来测序时类似大小的同一类型的文库可继续使用此上样浓度。 更多信息，请参考[Cluster Optimization Overview Guide](https://support.illumina.com/downloads/cluster-optimization-overview-guide-1000000071511.html)。 \ \ \
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