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 techsupport@illumina.com.

Last updated

© 2023 Illumina, Inc. All rights reserved. All trademarks are the property of Illumina, Inc. or their respective owners. Trademark information: illumina.com/company/legal.html. Privacy policy: illumina.com/company/legal/privacy.html