PhiX Indexed Control (1000 Cycle) Product Information

Background

The PhiX control library is a well-balanced, high diversity library that can be used as a positive control during Sequencing Runs on Illumina platforms. However, due to an average insert size of 375 bp and total library size of 500 bp, the current PhiX v3 product is not compatible with Reads longer than ~350 cycles.

The new PhiX Indexed Control (1000 Cycle) formulation addresses this by increasing the insert length, enabling users to spike-in PhiX control when performing high cycle count Runs.

Technical Details

• Insert Size

• The PhiX Indexed Control (1000 Cycle) formulation has an average insert size of 700 basepairs (bp)

• The total library length is approximately 825 bp on average

• Indexing Strategy

• The PhiX Indexed Control (1000 Cycle) library leverages a 10 bp, dual-index strategy and is a pool of 5 index combinations

• This provides additional diversity and color balance in Index Reads when spiked into low-plexity library pools

• The index combinations do not overlap with any current Illumina or IDT index combinations

• For other Third Party Libraries, please review manufacturer's Library Preparation information for potential overlapping indexes before using the PhiX Indexed Control (1000 Cycle) library

• (Optional) Demultiplexing PhiX Indexed Control

• Adding the indexed PhiX libraries to the Sample Sheet for demultiplexing is not required.

• If the indexed PhiX libraries are not added to the Sample Sheet, the reads sort to the Undetermined FASTQ files, similar to un-indexed PhiX v3 Control.

• If required, add the PhiX Sample ID and Index combinations in Table 1 to the Sample Sheet to generate FASTQ files for the individual PhiX libraries within the pool.

Table 1: PhiX Indexed Control (1000 Cycles) Index Sequences

PhiX Sample ID	i7 Bases for Sample Sheet	i5 Bases for Sample Sheet (Forward)	i5 Bases for Sample Sheet (Rev. Complement)
PhiX-Index1	CGACCTAACG	ATACGCCGGC	GCCGGCGTAT
PhiX-Index2	TAGTTCGGTA	CTGTCACTTA	TAAGTGACAG
PhiX-Index3	ACCGGCCGTA	GGCGCCATTG	CAATGGCGCC
PhiX-Index4	AATCACCAGC	CAGTAATTAC	GTAATTACTG
PhiX-Index5	AGCGAATTAG	GGCGATCAGA	TCTGATCGCC

• Loading Concentrations for the MiSeq i100 Series

  • See the MiSeq i100 Product Documentation for instructions on preparing PhiX spike-in controls.

  • See the Maximizing performance on the MiSeq i100 Series Technical Note for loading concentrations when setting up PhiX Control Run

  • For other applications, the appropriate loading concentration and spike-in value must be empirically determined.

• Platform Compatibility

  • The PhiX Indexed Control (1000 Cycle) is compatible with all Illumina sequencing platforms

  • Due to the larger total library length, the PhiX Indexed Control (1000 Cycle) may cluster less efficiently on some platforms, and optimal loading concentrations must be empirically determined

  • When spiking-in the PhiX Indexed Control (1000 Cycle) into libraries that are less than 900 bp average size, increase the concentration of PhiX Indexed Control (1000 Cycle) library to offset the larger insert size

For any feedback or questions regarding this article (Illumina Knowledge Article #9867), contact Illumina Technical Support [email protected].