Library quantification and quality control quick reference guide

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. This article summarizes the library quantification and quality control methods validated for libraries prepared with all current Illumina kits. Use the methods listed in the tables below to validate the final library before sequencing. Note that labs can choose validated alternative trace instruments, if desired.

Note: dsDNA = double stranded DNA; ssDNA = single stranded DNA.

DNA Kits - Research Use Only

Kit	Quantification	Quality control
TruSeq DNA Nano	dsDNA specific fluorometric method or qPCR	Fragment Analyzer or Bioanalyzer
TruSeq PCR Free	qPCR	Fragment Analyzer or Bioanalyzer
Illumina DNA Prep (M) Tagmentation	dsDNA specific fluorometric method	Fragment Analyzer or Bioanalyzer
Nextera XT	dsDNA specific fluorometric method or bead-based normalization	Bioanalzyer
Illumina DNA Prep with Enrchment (S) Tagmentation	dsDNA specific fluorometric method	Bioanalzyer
Illumina DNA PCR Free Tagmentation	ssDNA specific fluorometric method or qPCR	not recommended
TruSight Tumor 15	dsDNA specific fluorometric method	agarose gel or Bioanalzyer
AmpliSeq for Illumina (DNA panels)	dsDNA specific fluorometric method, qPCR, Fragment Analyzer, or Bioanalyzer	Fragment Analyzer or Bioanalyzer
TruSight Myeloid	bead-based normalization	agarose gel or Bioanalzyer
TruSeq ChIP	qPCR	Bioanalzyer

RNA Kits - Research Use Only

Kit	Quantification	Quality control
TruSeq RNA v2	qPCR	Fragment Analyzer or Bioanalyzer
TruSeq Stranded mRNA	qPCR	Fragment Analyzer or Bioanalyzer
TruSeq Stranded Total RNA	qPCR	Fragment Analyzer or Bioanalyzer
TruSeq RNA Exome	qPCR	Fragment Analyzer or Bioanalyzer
Illumina Stranded mRNA Prep, Ligation	dsDNA specific fluorometric method; qPCR for the NextSeq 1000/2000	Bioanalzyer or TapeStation
Illumina Stranded Total RNA Prep, Ligation	dsDNA specific fluorometric method; qPCR for the NextSeq 1000/2000	Bioanalzyer or TapeStation
Illumina RNA Prep with Enrichment (L) Tagmentation	dsDNA specific fluorometric method	Bioanalyzer
TruSeq Small RNA	Bioanalyzer	Bioanalyzer
AmpliSeq for Illumina (RNA panels)	dsDNA specific fluorometric method, qPCR, Fragment Analyzer, or Bioanalyzer	Fragment Analyzer or Bioanalyzer

DNA and RNA kits - Research Use Only

Kit	Quantification	Quality control
TruSight Tumor 170	bead-based normalization (optional, dsDNA specific fluorometric method before normalization)	not required
TruSight Oncology 500	bead-based normalization (optional, dsDNA specific fluorometric method before normalization)	not required
TruSight Oncology 500 High Throughput	bead-based normalization (optional, dsDNA specific fluorometric method before normalization)	not required
TruSight Oncology 500 ctDNA	bead-based normalization (optional, dsDNA specific fluorometric method before normalization)	not required

Notes:

• Fluorometric method = a double strand DNA-specific fluorescent dye method, such as Qubit, PicoGreen, or AccuClear.

• Additional information on qPCR quantification can be found in the Sequencing Library qPCR Quantification guide.

• Not Needed = quality control is not needed with bead-based normalized libraries.

For any feedback or questions regarding this article (Illumina Knowledge Article #1247), contact Illumina Technical Support [e-mail].