# 将定制引物掺入到Illumina测序引物中 \
在Illumina测序平台上利用定制引物对制备好的文库进行测序时，需要对其进行优化，以保证测序正常进行。当定制样本与PhiX或其它Illumina标准文库混合测序时，需要将定制引物与Illumina引物合并，或者将定制引物掺入到Illumina引物中。Illumina无法保证定制引物是否作用或者兼容，需要客户自己验证这些引物是可以在特定平台上进行测序。当使用定制测序引物时，最关键是需要了解基于平台推荐的装载浓度，总的体积，装载位置以及 [引物退火温度](https://support.illumina.com/bulletins/2016/10/considerations-when-migrating-nonillumina-libraries-between-sequencing-platforms.html)。该技术文档为如何将定制引物掺入到Illumina测序引物中提供了相关信息。\ **将定制引物掺入到Illumina引物中** * 下面这些表格分别提供了针对不同平台定制引物的试剂槽位置，总体积以及终浓度。 * 基于试剂槽中定制引物的终浓度，计算加入到Illumina引物试剂槽位置的定制引物体积\*。 * 掺入定制引物后，将吸液器体积调至总体积的一半，用吸液器缓慢上下吹打溶液5次，使之充分混匀\*\*。 * 对于MiSeq、MiniSeq、NextSeq500/550和NovaSeq6000平台，在sample sheet或者程序设置时，不要勾选 “custom primer” 选项。 \*可以用(C1)(V1) = (C2)(V2)等式计算需要掺入多少定制引物到对应孔里: C1=您定制引物的浓度（当使用高浓度引物储存液时，例如100uM，相对于最终的体积，额外增加的体积非常小，可以忽略） V1=掺入定制引物的体积 C2=参考下面表格中推荐的定制引物的终浓度 V2=参考下面表格中Illumina引物的总体积 例如: (100uM \* V1) = (0.5uM \* 680uL) V1 = 3.4 µL \*\*MiSeq、NextSeq和MiniSeq试剂槽：为了提高Illumina测序引物体积测定以及定制引物掺入和混匀的成功率，建议使用较细的枪头或者较大体积的低浓度定制引物。 **重要说明**: 以下指导资料是基于目前引物的体积。为了确保准确性，在进行操作前，请先测定试剂槽中引物的总体积。 **iSeq 100** 随着iSeq 控制软件3.0的发布，现在iSeq100也可以用定制引物测序。由于iSeq试剂卡盒的构造原因，我们无法直接将定制引物提前加入到Illumina 引物中。 不同于将定制引物直接混入试剂卡盒的引物孔中，iSeq 100定制引物测序需要用到NextSeq 1000/2000 Custom Primer试剂盒，并且该试剂盒配置是兼容iSeq 100的。定制引物需要先添加到适当Illumina引物中，然后按照iSeq 100定制引物流程添加到试剂卡盒中。对于NextSeq 1000/2000 Custom Primer试剂盒的说明和货号，请查阅 [NextSeq 1000/2000 Custom Primers Guide](https://support-docs.illumina.com/IN/NextSeq10002000/Content/IN/NextSeq2000_1000/CustomPrimers_Intro_fNS_m2000.htm). **重要说明**：NextSeq 1000/2000定制引物指南提供了针对NextSeq 1000/2000的引物添加说明。对于iSeq100定制引物的添加请参阅以下说明。 | Kit version | Illumina Primer (name) |

Cartridge
Position

|

Total
Volume (µL)

|

Custom primer final
concentration (µM)

| | ------------------- | ---------------------- | ---------------------------- | --------------------------- | ------------------------------------------------ | | i1 v2 | Read 1 (HP21) | NA | 140 | 0.3 | | Index 1 (i7) (BP14) | NA | 140 | 0.6 | | | Index 2 (i5) (BP14) | NA | 140 | 0.6 | | | Read 2 (HP21) | NA | 140 | 0.3 | | **MiniSeq** 由于MiniSeq上独特的双标签测序流程，定制引物与Illumina引物的组合有一定限制。需要注意定制引物的从属关系，请参考相应[文档](http://support.illumina.com/downloads/miniseq-custom-primers-guide-1000000002700.html)。 | Kit version | Illumina Primer (name) |

Cartridge
Position

|

Total
Volume (µL)

|

Custom primer final
concentration (µM)

| | ------------------------------------ | ---------------------- | ---------------------------- | --------------------------- | ------------------------------------------------ | |

Rapid Run
100 Cycles

| Read 1 (BP10) | 24 | 590 | 0.3 | | Read 2 (BP11) | 25 | 530 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 28 | 740 | 0.3 | | |

High Output
75 Cycles

| Read 1 (BP10) | 24 | 550 | 0.3 | | Read 2 (BP11) | 25 | 610 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 28 | 820 | 0.3 | | |

High Output
150 Cycles

| Read 1 (BP10) | 24 | 550 | 0.3 | | Read 2 (BP11) | 25 | 610 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 28 | 857 | 0.3 | | |

High Output
300 Cycles

| Read 1 (BP10) | 24 | 550 | 0.3 | | Read 2 (BP11) | 25 | 610 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 28 | 820 | 0.3 | | |

Mid Output
300 Cycles

| Read 1 (BP10) | 24 | 550 | 0.3 | | Read 2 (BP11) | 25 | 610 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 28 | 820 | 0.3 | | **MiSeq** | Kit version | Illumina Primer (name) |

Cartridge
Position

|

Total
Volume (µL)

|

Custom primer final
concentration (µM)

| | --------------------- | ---------------------- | ---------------------------- | --------------------------- | ------------------------------------------------ | | v2 and v3 | Read 1 (HP10) | 12 | 680 | 0.5 | | Index 1 (i7) (HP12)\* | 13 | 680 | 0.5 | | | Read 2 (HP11) | 14 | 680 | 0.5 | | \* 由于模板使用流动槽(Flowcell)表面偶联的P5引物为测序引物，因此无法使用Index2(i5)定制引物。 **MiSeq i100 Series** 对于Miseq i100测序的定制引物添加请参考 [Spiking custom primers into the Illumina sequencing primers for MiSeq i100 Series](https://knowledge.illumina.com/instrumentation/miseq-i100-series/instrumentation-miseq-i100-series-reference_material-list/000009551)。 **NextSeq 500/550** 由于NextSeq上独特的双标签测序流程，定制引物与Illumina引物的组合有一定限制。需要注意定制引物的从属关系，请参考相应[文档 ](https://support.illumina.com/downloads/nextseq-500-custom-primers-guide-15057456.html)。 | Kit version | Illumina Primer (name) |

Cartridge
Position

|

Total
Volume (mL)

|

Custom primer final
concentration (µM)

| | ------------------------------------ | ---------------------- | ---------------------------- | --------------------------- | ------------------------------------------------ | |

High Output
75 cycles

| Read 1 (BP10) | 20 | 1.72 | 0.3 | | Read 2 (BP11) | 21 | 1.98 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 22 | 2.83 | 0.3 | | |

High Output
150 Cycles

| Read 1 (BP10) | 20 | 1.73 | 0.3 | | Read 2 (BP11) | 21 | 1.98 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 22 | 2.83 | 0.3 | | |

High Output
300 Cycles

| Read 1 (BP10) | 20 | 1.73 | 0.3 | | Read 2 (BP11) | 21 | 1.98 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 22 | 2.83 | 0.3 | | |

Mid Output
150 Cycles

| Read 1 (BP10) | 20 | 1.34 | 0.3 | | Read 2 (BP11) | 21 | 1.51 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 22 | 2.09 | 0.3 | | |

Mid Output
300 Cycles

| Read 1 (BP10) | 20 | 1.33 | 0.3 | | Read 2 (BP11) | 21 | 1.52 | 0.3 | | | Index 1 (i7) and Index 2 (i5) (BP14) | 22 | 2.09 | 0.3 | | **NextSeq 1000/2000** 由于NextSeq 1000/2000试剂卡盒的构造原因，无法直接将定制引物加入到Illumina测序引物中。 如果在测序文库中有Phix需要用到Illumina测序引物，就需要用到NextSeq 1000/2000 Custom Primer试剂盒。具体请参阅[NextSeq 1000/2000 Custom Primers Guide](https://support-docs.illumina.com/IN/NextSeq10002000/Content/IN/NextSeq2000_1000/CustomPrimers_Intro_fNS_m2000.htm)。 **NovaSeq 6000 v1.5 Consumables** 由于NovaSeq 6000上的引物设计和双标签测序流程，定制引物与Illumina引物的组合有一定限制。需要注意定制引物的从属关系，请参考[文档](https://support.illumina.com/downloads/novaseq-custom-primers-guide-1000000022266.html)。 | Kit version | Illumina Primer (name) |

Cartridge
Position

|

Total
Volume (mL)

|

Custom primer final
concentration (µM)

| | -------------------------------------- | ---------------------- | ---------------------------- | --------------------------- | ------------------------------------------------ | |

SP
100/200/300/500 cycles

| Read 1 (VP10) | 24 | 4 | 0.3 | | Index 1 (i7) and Index 2 (i5) (VP14) | 23 | 5 | 0.3 | | | Read 2 (VP11) | 13 | 2 | 0.3 | | |

S1 and S2
100/200/300 cycles

| Read 1 (VP10) | 24 | 4 | 0.3 | | Index 1 (i7) and Index 2 (i5) (VP14) | 23 | 5 | 0.3 | | | Read 2 (VP11) | 13 | 2 | 0.3 | | |

S4
200/300 cycles

| Read 1 (VP10) | 24 | 7.3 | 0.3 | | Index 1 (i7) and Index 2 (i5) (VP14) | 23 | 5 | 0.3 | | | Read 2 (VP11) | 13 | 3.5 | 0.3 | | **NovaSeq X/X Plus** NovaSeq X/X Plus 采用了两种引物溶液，它们分别包含了Read1 和 Read2以及Index1和Index2 所需的引物。如果仅为特定的Reads需要定制引物测序，请将高浓度的定制引物掺入到需要定制引物的相应孔中。在掺入定制引物时，**无需更改**运行设置，也无需使用定制配方（custom recipe）。 该表格列出了孔位信息、Illumina 引物体积以及建议添加的定制引物（CP）体积（单位为微升，µL），**假设所使用的定制引物储备液的初始浓度为 100 微摩尔（µM）。** ![](/files/26vIVX5d5iUeitIZhuX9) **使用定制引物替代Illumina引物** 如果仅需要定制引物，可以用定制引物替代Illumina引物。当仅使用定制引物时，设置运行程序的说明，可以参考以下文档： * [MiniSeq System Custom Primers Guide](http://support.illumina.com/downloads/miniseq-custom-primers-guide-1000000002700.html) * [MiSeq System Custom Primers Guide](http://support.illumina.com/downloads/using_customprimers_miseq_15041638.html) * [NextSeq 500/550 System Custom Primers Guide](http://support.illumina.com/downloads/nextseq-500-custom-primers-guide-15057456.html) * [NextSeq 1000/2000 Product Documentation - Custom Primers](https://support-docs.illumina.com/IN/NextSeq10002000/Content/IN/NextSeq2000_1000/CustomPrimers_Intro_fNS_m2000.htm) * [HiSeq System Custom Primers Guide](http://support.illumina.com/downloads/hiseq-using-custom-primers-reference-guide-15061846.html) * [NovaSeq Series Custom Primers Guide](https://support.illumina.com/downloads/novaseq-custom-primers-guide-1000000022266.html) * [iSeq 100 Sequencing System - Custom Primers](https://support-docs.illumina.com/IN/iSeq100/Content/IN/iSeq/CustomPrimers-Intro.htm#CustPrimers-Intro) * [NovaSeq X Plus Product Documentation - Custom Primers](https://support-docs.illumina.com/IN/NovaSeqX/Content/IN/NovaSeqX/CustomPrimers.htm) 关于各个平台标签测序的更多信息，请参考文档[Indexed Sequencing Overview Guide](https://support.illumina.com/downloads/indexed-sequencing-overview-15057455.html). \ \ \
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