PhiRx™ Indexed Control
PhiRx™ Indexed Control is a dual-indexed library derived from the phiX174 genome, specifically engineered to optimize the performance of XLEAP-SBS™ chemistry. It serves as a seamless spike-in to ensure signal uniformity and high data quality on two-color Illumina platforms, such as the NextSeq™ 1000/2000 and NovaSeq™ X.
- 10 bp i5 and i7 indexes with mixed bases: Provides high base diversity, automatically meeting color balancing requirements on supported platforms (patent-pending).
- Color balancing optimization: Specifically designed for the sensitive XLEAP SBS chemistry, enabling as little as 5% spike-in to correct base calling issues in libraries with low-diversity indexes.
- High diversity for low-plexity samples: Randomized inserts with 45% GC content improve sequencing accuracy for low-diversity libraries.
- Reliable quality control: Derived from the well-characterized phiX174 genome, providing robust calibration to support sequencing accuracy and performance.
| Feature | Specification |
| Genome source | phiX174 |
| Index Type | 10 bp i5 and i7 dual indexes |
| GC Content | ~45% (Randomized inserts) |
| Compatibility | Illumina XLEAP-SBS (NextSeq 1000/2000, NovaSeq X) |
| Recommended Loading | 5% spike-in |
| Article Number | Description |
| 300184 | PhiRx Indexed Control, 10μL tube at 10nM concentration |
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Enhanced Demultiplexing
Solving Color-Balancing Challenges in Low-Plexity Sequencing
Standard controls often struggle with the sensitive color-balancing requirements of modern XLEAP-SBS chemistry, particularly when processing small batches or low-diversity libraries. PhiRx™ addresses this with patent-pending 10 bp indexes featuring mixed bases. This design provides the necessary base diversity to meet platform requirements automatically, effectively correcting base-calling issues and recovering data that would otherwise be lost as undetermined reads.
| Spike-in Sequencing Control | % PhiX Aligned | % Undetermined Reads (Excluding PhiX) | % One-mismatch |
| seqWell PhiRx Indexed Control | 5.22 | 1.82 | 4.15 |
| Illumina PhiX Control V3 | 6.27 | 53.25 | 100.0 |
To obtain this data, a 96-plex library prepared using ExpressPlex 2.0 was sequenced on a NextSeq 2000 with 5% spike-in of Illumina PhiX Control V3 or PhiRx from seqWell.
Implementation & Sequencing Optimization
Technical guidance on using PhiRx Indexed Control for color balancing, library preparation, and performance monitoring on Illumina platforms.
PhiRx Indexed Control should be added to runs for color-balancing both the reads and barcodes. Other sequencing spike-ins should not be required for most use cases.
PhiRx Indexed Control is a non-denatured, double-stranded library that is ready to be mixed with non denatured target libraries. For applications requiring a denatured format, PhiRx Indexed Control can also be denatured following standard procedures and mixed exclusively with other denatured libraries.
Please check Illumina recommendations specific to your sequencing platform and analysis software version to determine the appropriate PhiRx Indexed Control spike-in percentage.
Recommended Spike-In Ratios by Application:
- For Sequencing Quality Monitoring: Add 1–2% of PhiRx Indexed Control to the target library.
- For Color Balancing of Index Reads with XLEAP-SBS: Begin with 5–10% PhiRx Indexed Control. Per Illumina's recommendation for their PhiX Sequencing Control V3, using a higher percentage of PhiRx (up to 40%) may improve performance, especially with XLEAP-SBS 600 cycle kits.
- For Low-Diversity Libraries (Color Balancing of Insert Reads): Start with 15–20% PhiRx Indexed Control and adjust upward if needed to enhance sequencing performance.
Follow Illumina’s recommendations for diluting and loading PhiX Sequencing Control V3. Use the buffers recommended by Illumina for diluting and dentaturing. If diluting below 1 nM, store and use active aliquots for up to 2 weeks at 4°C. Aliquots not currently in use should be kept at -20°C for long-term storage.
Successful use of PhiRx Indexed Control is defined by seeing a decrease in the %unassigned reads minus the %PhiX aligned in your sequencing run metrics.
PhiRx Indexed Control is stable for at least 8 freeze-thaw cycles.
The mean fragment size is ~476bp.
PhiRx Indexed Control uses dual 10 bp indexes (10 bp i7 + 10 bp i5), with each position composed of randomly mixed bases. This design results in a very low probability of any specific ten-base index sequence matching a sample barcode.
When ExpressPlex kits are used, the mismatch rate is approximately 0.0001%, meaning about 1 mismatched read per 1 million reads when allowing for one mismatch per index read during demultiplexing. If unique dual indexes (UDIs) are used, the mismatch rate is expected to be even lower.