Library quantification and quality control quick reference guide

01/29/19


Accurate quantification and proper quality check of next-generation sequencing libraries is key to a successful sequencing run. Different methods for quantification and quality control are recommended 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 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
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
Nextera DNA Flex for Enrichment QuBit BioAnalyzer
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
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
Ampliseq (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

 

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

 

Notes:

  • Fluorometric method = a dsDNA-specific fluorescent dye method, such as QuBit, PicoGreen, and AccuClear
  • Additional information on qPCR quantification can be found in the qPCR guide
  • Not Needed = quality control is not needed with bead-based normalized libraries