Qualification/QC FAQ for Illumina DNA PCR Free libraries

What is the typical library size distribution for Illumina DNA PCR-Free libraries? Median sequenced insert size is ~450 bp based on sequencing alignment. The final library size cannot be determined as ssDNA libraries will not resolve on traces such as Bioanalyzer, Fragment Analyzer, or TapeStation.

What is the insert size for Illumina PCR-Free libraries?

• Median insert size is ~450 bp on NovaSeq 6000. Patterned flow cells typically provide insert sizes around 450 bp. For non-patterned flow cells, insert sizes tend to be approximately 100 bp shorter.

• Due to the different mechanism of clustering between non-patterned (MiniSeq, MiSeq, and NextSeq 500/550) and patterned (NovaSeq 6000, NextSeq 1000/2000, NovaSeq X Series) flow cells, variation in the median insert size of sequenced libraries may be observed between systems. These size differences are not reflective of library preparation. See app note Optimal loading concentrations for Illumina DNA PCR-Free libraries for details.

How do the libraries end up as single stranded libraries when double-stranded DNA is used as an input? Does this happen when the BLT-PF beads are added? There is an elution step from BLT-PF beads after adapter ligation with diluted HP3 (NaOH) which is responsible for libraries becoming DNA single-stranded.

Can an RNA BioAnalyzer/Fragment Analyzer/TapeStation trace be run to visualize the libraries, since RNA is also single-stranded? Is an example trace available for reference?

• No, the trace will not be visible.

Are final library adapters forked, as in TruSeq DNA PCR-Free libraries? No, unlike TruSeq DNA PCR-Free, Illumina DNA PCR-Free yields single stranded DNA libraries, and so there are no double stranded regions at the conclusion of the protocol from which a "fork" would be present.

Is there a way to evaluate library size to verify it is as expected before sequencing? No.

For any feedback or questions regarding this article (Illumina Knowledge Article #3478), contact Illumina Technical Support [e-mail].