Samples per sequencing run and coverage FAQ for Illumina DNA PCR Free

Will both library strands be sequenced, or only one? Both strands of gDNA will be sequenced.

What is the minimum and maximum number of samples that can be pooled in one sequencing run? Due to the differing needs for sequencing depth and different sample types used, there is no single answer; refer to the Sequencing Coverage Calculator.

What is the recommended sequencing depth/number of reads per sample? This is highly dependent on data use case/data needs. See the app note High-performance whole-genome sequencing with Illumina DNA PCR-Free Prep, Tagmentation for more detail.

Will Illumina DNA PCR-Free require higher sequencing depth in comparison to TruSeq DNA PCR-Free libraries? Illumina DNA PCR-Free generate data comparable to TruSeq DNA PCR-Free.

What coverage level of each sample is recommended for maximum accuracy of genome assembly and variant calling? This is a multi-faceted question and depends on specific data needs. See app note High-performance whole-genome sequencing with Illumina DNA PCR-Free Prep, Tagmentation and the product data sheet for more information.

Is there a pooling calculator for these libraries? See the Illumina Pooling Calculator.

What is the recommended loading concentration?

• Libraries created with the standard input workflow can be run with the NovaSeq 6000 Xp or standard workflows. Libraries created with the low input workflow should use the NovaSeq 6000 Xp workflow only. Loading concentrations for NovaSeq 6000 are in the Illumina DNA PCR-Free Reference Guide.

• For non-NovaSeq instruments, refer to the application note Optimal loading concentrations for Illumina DNA PCR-Free libraries for suggested loading concentrations.

For any feedback or questions regarding this article (Illumina Knowledge Article #3442), contact Illumina Technical Support [e-mail].