# 如何排查MiSeq平台上的第一个循环报错的原因？ \
MiSeq平台在第一个循环拍照时发生报错通常表明簇信号强度不够，仪器不能找到最佳的聚焦平面。这可能是由于簇密度过高、过低或簇生成失败造成的。 可能的报错信息包括： * Best focus not found * Best focus is too near the edge of range * No usable signal found, it is possible clustering has failed * Through-focus peak did not exceed SNR threshold * Z Motor attempt to move outside soft limits * New in MiSeq Control Software (MCS) v4.0: ThroughFocusScan CoarseFocus failed to find focus, and there is no fallback position. ![](/files/sRRN38BLIMlwyYPo2WVD) 第一个循环报错可能由文库、运行设置、仪器或试剂问题引起。 可能的仪器原因包括： * 参与簇生成或者第一个碱基化学反应试剂的液流问题 * 流动槽温控问题 * 光学系统问题（这种情况非常少） 可能的文库和试剂原因包括： * 使用了过期或者储存条件不当的试剂 * 文库设计或者文库质量/定量问题 * 由于文库设计问题导致Read1引物杂交不好 * 使用了不合适的引物 * 簇密度过高或者过低 * NaOH的pH值 * 清洗槽的污染 第一个循环的错误表明仪器在第一个循环未能识别到足够信号，无法计算出flow cell上的适当焦点位置。MiSeq测序仪在首轮成像后出现的错误可能意味着簇强度不足，导致仪器无法找到最佳聚焦平面。 如果聚焦失败发生在测序过程中 (例如index的第一个循环或者Read1或Read4的中途) ，请搜索illumina knowledge Bases中的技术文档[Troubleshooting best focus errors occurring mid-run on the MiSeq](https://knowledge.illumina.com/instrumentation/miseq/instrumentation-miseq-troubleshooting-list/000001849), [Troubleshooting Z motor errors occurring mid-run ](https://knowledge.illumina.com/instrumentation/miseq/instrumentation-miseq-troubleshooting-list/000001850)和 [Troubleshooting No Intensity for Index Read on MiSeq](https://knowledge.illumina.com/instrumentation/miseq/instrumentation-miseq-troubleshooting-list/000001461)进行问题排查。 **故障排查步骤：** 首先可以按照[MiSeq System Guide](https://support.illumina.com/sequencing/sequencing_instruments/miseq/documentation.html)对仪器进行系统检查，确保仪器的流路、温度以及机械移动系统正常运行： 1. 按照仪器提示执行post run wash (运行后清洗) 2. 按照使用手册，重启机器 3. 去Manage Instrument（管理仪器）界面，点击System Check（系统检查） 4. 选择所有的运动测试，试剂管路冲洗，温度梯度以及液量测试。 当系统检查完成，点击屏幕左下方的Show Details（展开细节）按钮，得到检查结果的完整信息。如果是PR2以外的任何液量测试或者其它测试失败，请发邮件到[chinasupport@illumina.com](mailto:techsupport@illumina.com)去获得更多技术支持。 如果系统检查通过，则要考虑报错可能是由文库或者试剂问题导致的。我们需要分析以下常见问题： * 检查试剂盒的有效日期以及正确储存条件。 * 确保文库设计适合在Illumina平台测序。 * 利用Illumina推荐的方法，检查文库质量和浓度。 * 确保自定义引物适合MiSeq 测序平台 * 确保自定义引物加入到试剂夹盒正确的孔位中（如果加入到已有引物的试剂孔中，sample sheet不需要修改，如果加入到自定义引物的试剂孔中，则sample sheet需要选择自定义引物） * NaOH是新鲜稀释的且pH值大于12.5 如果文库或试剂没有明显问题，在文库中掺入20% PhiX重新测序。PhiX可以作为下一次测序簇生成的阳性对照，进而判断之前的错误是否是由文库问题导致的。 如果后续运行在第一个循环再次因错误停止，可以发邮件到Illumina技术支持邮箱[chinasupport@illumina.com](mailto:techsupport@illumina.com)获得更多技术支持。 更多资源链接: [Denature and Dilute protocol generator](https://support-docs.illumina.com/IN/dnd-wizard/Content/dnd/landing.htm) [MiSeq System Custom Primers Guide](https://support.illumina.com/downloads/miseq-system-custom-primers-guide-15041638.html) **更多信息，请搜索 Knowledge Base中以下文档:** [Spiking custom primers into the Illumina sequencing primers](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000001542) [How to achieve more consistent cluster density on Illumina sequencing platforms](https://knowledge.illumina.com/instrumentation/general/instrumentation-general-reference_material-list/000001481) [How to perform a System Check on the MiSeq](https://knowledge.illumina.com/instrumentation/miseq/instrumentation-miseq-reference_material-list/000001777) [How To Power Cycle the MiSeq Video](https://knowledge.illumina.com/instrumentation/miseq/instrumentation-miseq-reference_material-list/000005871) [Troubleshooting best focus errors occurring mid-run on the MiSeq](https://knowledge.illumina.com/instrumentation/miseq/instrumentation-miseq-troubleshooting-list/000001849) [Troubleshooting Z motor errors occurring mid-run on the MiSeq](https://knowledge.illumina.com/instrumentation/miseq/instrumentation-miseq-troubleshooting-list/000001850) [Troubleshooting No Intensity for Index Read on MiSeq](https://knowledge.illumina.com/instrumentation/miseq/instrumentation-miseq-troubleshooting-list/000001461) \ \ \
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