文库定量与质量控制简明指引

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。