Workflow Optimization by Modulating SMB4 Volume for Illumina DNA Prep with Exome 2.5 Enrichment v2

The standard volume for SMB4 usage with Illumina DNA Prep with Exome 2.5 Enrichment v2 is 200 µl, down from 250 µl used in v1 of the kit. This change contributes to improvements in overall coverage of GC targets, and is the recommended volume for customers to use. Though if customers see lower than desired coverage of critical high %GC regions, this volume may be reduced to as low as 100 µl to try to improve coverage of these regions. However, at volumes less than 200 µl, SNV and indel recall rates may be negatively impacted.

In an experiment, 50 ng of NA12878 Coriell DNA was used as input, and prepared into libraries following the Illumina DNA Prep with Exome 2.5 Enrichment v2 protocol. Probe capture was performed with either 100 µl or 200 µl of SMB4. The final libraries were sequenced on a NovaSeq 6000 S4 flow cell using the XP workflow, at 2x101 bp read lengths. Data were downsampled to 50 million total reads using DRAGEN FASTQ Toolkit v1.3.1, and Picard metrics and analysis results were generated with DRAGEN Enrichment v4.3.13. Data was compared to VCAT v1:4.1.0-NA12878 NIST GIAB (Genome in a Bottle) truth set.

The effects of using either 100 µl of SMB4 or 200 µl of SMB4 are shown in Table 1. Overall, 200 µl SMB4 is the best starting point; labs can see slightly better GC coverage with a reduced volume (green), but at the cost of higher AT dropout and impacts on variant calling (red).

Table 1. Impact on enrichment metrics for Illumina DNA Prep with Exome 2.5 Enrichment v2 libraries prepared with either 100 µl or 200 µl SMB4. Note that "Hybrid Selection Library Size" is an analysis done by Picard which is an estimate of the number of unique DNA molecules present in the library after capture.

Demo data will be available on BaseSpace Sequence Hub (BSSH) to assist labs in evaluating the change in coverage with 100 µl vs 200 µl SMB4. However, as results can vary based on the specific sample and loci of interest, customers should perform pilot experiments and optimize the volume of SMB4 for their regions of interest, if choosing to use < 200 µl, the recommended volume of SMB4.

For any feedback or questions regarding this article (Illumina Knowledge Article #10120), contact Illumina Technical Support [email protected].