05/17/21
测序文库精确的定量与准确的质量检测对于二代测序成功是十分关键的。对于不同测序文库试剂盒，我们推荐使用不同的定量和质量控制的方法。在本篇技术文档中，我们总结了目前所有Illlumina建库试剂盒的文库定量与质量控制的方法。在测序前，利用下表中的方法对最终文库进行鉴定。
DNA试剂盒-仅用于科研
| 试剂盒 | 定量 | 质量控制 |
|---|---|---|
| TruSeq Nano DNA | 荧光方法或qPCR | BioAnalyzer 或 Fragment Analyzer |
| TruSeq PCR Free | qPCR | BioAnalyzer 或 Fragment Analyzer |
| Illumina DNA Prep, (M) Tagmentation (formerly Nextera DNA Flex) |
荧光方法(Qubit/PicoGreen) | BioAnalyzer 或 Fragment Analyzer |
| Nextera XT | 荧光方法或基于磁珠均一化 | BioAnalyzer (当使用荧光方法时需要；基于磁珠进行均一化时为可选) |
| Nextera Mate Pair | qPCR/BioAnalyzer | BioAnalyzer或琼脂糖凝胶 |
| Illumina DNA Prep with Enrichment, (S) Tagmentation (formerly Nextera DNA Flex for Enrichment) |
Qubit | BioAnalyzer |
| Illumina DNA PCR-Free Prep, Tagmentation | Qubit ssDNA或基于投入量的qPCR, 具体细节可参照 Reference Guide | N/A (最终文库为单链DNA) |
| TruSeq Exome | 荧光方法或qPCR | BioAnalyzer 或 Fragment Analyzer |
| Nextera DNA Exome | 荧光方法或qPCR | BioAnalyzer 或 Fragment Analyzer |
| Nextera Rapid Capture Custom Enrichment | 荧光方法或qPCR | BioAnalyzer |
| TruSight Inherited Disease | 荧光方法或qPCR | BioAnalyzer |
| TruSight Tumor 15 | 荧光方法(AccuClear/PicoGreen/Qubit) | BioAnalyzer或琼脂糖凝胶 |
| TruSight Tumor 26 (TruSight Tumor) | BioAnalyzer | 琼脂糖凝胶/BioAnalyzer |
| Ampliseq (DNA Panels) | BioAnalyzer/Fragment Analyzer/qPCR/Qubit/AccuClear/QuantIT | BioAnalyzer 或 Fragment Analyzer |
| TruSight Myeloid | 基于磁珠均一化 | BioAnalyzer或琼脂糖凝胶 |
| TruSeq Amplicon Cancer Panel | 基于磁珠均一化 | BioAnalyzer或琼脂糖凝胶 |
| TruSeq Bovine Parentage Sequencing Panel | 基于磁珠均一化 | BioAnalyzer/Fragment Analyzer/Agarose Gel |
| TruSeq ChIP | qPCR | BioAnalyzer |
| TruSeq Methyl Capture EPIC Library Prep Kit | Qubit | BioAnalyzer |
RNA 试剂盒-仅用于科研
| 试剂盒 | 定量 | 质量控制 |
|---|---|---|
| TruSeq RNA v2 | qPCR | BioAnalyzer 或 Fragment Analyzer |
| TruSeq Stranded mRNA | qPCR | BioAnalyzer 或 Fragment Analyzer |
| TruSeq Stranded Total RNA | qPCR | BioAnalyzer 或 Fragment Analyzer |
| TruSeq RNA Exome | qPCR/荧光方法 | BioAnalyzer 或 Fragment Analyzer |
| Illumina Stranded mRNA Prep, Ligation | Qubit | BioAnalyzer |
| Illumina Stranded Total RNA Prep with Ribo-Zero Plus | Qubit | BioAnalyzer |
| Illumina RNA Prep with Enrichment, (L) Tagmentation | Qubit | BioAnalyzer |
| TruSight RNA Pan-Cancer | qPCR | BioAnalyzer 或 Fragment Analyzer |
| TruSight RNA Fusion | qPCR | BioAnalyzer 或 Fragment Analyzer |
| TruSeq Targeted RNA Expression | BioAnalyzer 或 Fragment Analyzer | BioAnalyzer 或 Fragment Analyzer |
| TruSeq Small RNA | BioAnalyzer | BioAnalyzer |
| Ampiseq (RNA Panels) | BioAnalyzer/Fragment Analyzer/qPCR/Qubit/AccuClear/QuantIT | BioAnalyzer 或 Fragment Analyzer |
| SureCell WTA 3' | BioAnalyzer | BioAnalyzer |
DNA和RNA试剂盒-仅用于科研
| 试剂盒 | 定量 | 质量控制 |
|---|---|---|
| TruSight Tumor 170 | Fluorometric Method/基于磁珠均一化 | 不需要 |
| TruSight Oncology 500 | AccuClear/基于磁珠均一化 | 不需要 |
| TruSight Oncology 500 High Throughput | AccuClear/基于磁珠均一化 | 不需要 |
| TruSight Oncology 500 ctDNA | AccuClear/基于磁珠均一化 | 不需要 |
分子诊断试剂盒
| Kits | 定量 | 质量控制 |
|---|---|---|
| Cystic Fibrosis 139-Variant | 基于磁珠均一化 | 不需要 |
| Cystic Fibrosis Clinical Sequencing | 基于磁珠均一化 | 不需要 |
| TruSeq Custom Amplicon Dx | 基于磁珠均一化 | 不需要 |
注意：
· 荧光方法为双链DNA特异荧光染料方法，例如QuBit, PicoGreen和AccuClear。
· qPCR定量方法更多的信息可以参考qPCR guide。
· 基于磁珠进行均一化的文库不需要进行质量控制。