# Qualification/QC FAQ for Illumina DNA PCR Free libraries

p>**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](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/illumina-dna-pcr-free-loading-concentration-tech-note-770-2020-007/illumina-dna-pcr-free-loading-concentration-tech-note-770-2020-007.pdf) 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.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3478), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003478%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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