03/31/21
Accurate quantification and proper quality check of next-generation sequencing libraries is key to a successful sequencing run. There are different methods for quantification and quality control depending on the sequencing library kit being used. In this bulletin, we summarize the library quantification and quality control methods for libraries prepared with all current Illumina kits. Use the methods listed in the tables below to validate the final library before sequencing.
DNA Kits – Research Use Only
| Kits | Quantification | Quality Control |
|---|---|---|
| TruSeq Nano DNA | Fluorometric Method or qPCR | BioAnalyzer or Fragment Analyzer |
| TruSeq PCR Free | qPCR | BioAnalyzer or Fragment Analyzer |
|
Illumina DNA Prep, (M) Tagmentation (formerly Nextera DNA Flex) |
Fluorometric Method (Qubit/PicoGreen) | BioAnalyzer or Fragment Analyzer |
| Nextera XT | Fluorometric Method or Bead-based Normalization | BioAnalyzer (Needed when using Fluorometric Method; Optional when using Bead-based Normalization) |
| Nextera Mate Pair | qPCR/BioAnalyzer | BioAnalyzer or Agarose Gel |
|
Illumina DNA Prep with Enrichment, (S) Tagmentation (formerly Nextera DNA Flex for Enrichment) |
Qubit | BioAnalyzer |
| Illumina DNA PCR-Free Prep, Tagmentation | Qubit ssDNA or qPCR based on input amount, see Reference Guide for full details | N/A (final library is single stranded DNA) |
| TruSeq Exome | Fluorometric Method or qPCR | BioAnalyzer or Fragment Analyzer |
| Nextera DNA Exome | Fluorometric Method or qPCR | BioAnalyzer or Fragment Analyzer |
| Nextera Rapid Capture Custom Enrichment | Fluorometric Method or qPCR | BioAnalyzer |
| TruSight Inherited Disease | Fluorometric Method or qPCR | BioAnalyzer |
| TruSight Tumor 15 | Fluorometric Method (AccuClear/PicoGreen/Qubit) | BioAnalyzer or Agarose Gel |
| TruSight Tumor 26 (TruSight Tumor) | BioAnalyzer | Gel/BioAnalyzer |
| Ampliseq (DNA Panels) | BioAnalyzer/Fragment Analyzer/qPCR/Qubit/AccuClear/QuantIT | BioAnalyzer or Fragment Analyzer |
| TruSight Myeloid | Bead-based Normalization | BioAnalyzer or Agarose Gel |
| TruSeq Amplicon Cancer Panel | Bead-based Normalization | BioAnalyzer or Agarose Gel |
| TruSeq Bovine Parentage Sequencing Panel | Bead-based Normalization | BioAnalyzer/Fragment Analyzer/Agarose Gel |
| TruSeq ChIP | qPCR | BioAnalyzer |
| TruSeq Methyl Capture EPIC Library Prep Kit | Qubit | BioAnalyzer |
RNA Kits – Research Use Only
| Kits | Quantification | Quality Control |
|---|---|---|
| TruSeq RNA v2 | qPCR | BioAnalyzer or Fragment Analyzer |
| TruSeq Stranded mRNA | qPCR | BioAnalyzer or Fragment Analyzer |
| TruSeq Stranded Total RNA | qPCR | BioAnalyzer or Fragment Analyzer |
| TruSeq RNA Exome | qPCR/Fluorometric Method | BioAnalyzer or 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 or Fragment Analyzer |
| TruSight RNA Fusion | qPCR | BioAnalyzer or Fragment Analyzer |
| TruSeq Targeted RNA Expression | BioAnalyzer or Fragment Analyzer | BioAnalyzer or Fragment Analyzer |
| TruSeq Small RNA | BioAnalyzer | BioAnalyzer |
| Ampiseq (RNA Panels) | BioAnalyzer/Fragment Analyzer/qPCR/Qubit/AccuClear/QuantIT | BioAnalyzer or Fragment Analyzer |
| SureCell WTA 3' | BioAnalyzer | BioAnalyzer |
DNA and RNA kits - Research Use Only
| Kits | Quantification | Quality Control |
|---|---|---|
| TruSight Tumor 170 | Fluorometric Method/Bead-based Normalization | Not Needed |
| TruSight Oncology 500 | AccuClear/Bead-based Normalization | Not Needed |
| TruSight Oncology 500 High Throughput | AccuClear/Bead-based Normalization | Not Needed |
| TruSight Oncology 500 ctDNA | AccuClear/Bead-based Normalization | Not Needed |
Molecular Diagnostics Kits
| Kits | Quantification | Quality Control |
|---|---|---|
| Cystic Fibrosis 139-Variant | Bead-based Normalization | Not Needed |
| Cystic Fibrosis Clinical Sequencing | Bead-based Normalization | Not Needed |
| TruSeq Custom Amplicon Dx | Bead-based Normalization | Not Needed |
Notes: