Index Balance Checker

Lexogen Indices for Illumina (QuantSeq, SENSE, and Small RNA-Seq Kits)

The Index Balance Checker tool will help you select the optimal index combination for multiplexed sequencing for any number of samples. The app below displays the nucleotide and color balance of your chosen Lexogen i7 and i5 indices for QuantSeq/SENSE, and the SRi7 indices for the Small RNA-Seq Kit, and reports index balance relevant for all Illumina platforms (HiSeq, MiSeq, MiniSeq, NextSeq, and NovaSeq).

How to use the Index Balance Checker – Plan, Check, Multiplex!

  1. Select the kit size (8, 24, or 96 preps for SENSE and Small RNA-Seq Kits, and 24 or 96 preps for QuantSeq Kits).
  2. Select the Imaging Mode by clicking the button for the instrument type.
  3. Select the Index Set (i7/SRi7, i5, or i5rc).
  4. Select individual indices by clicking on the cells in the plate layout. Hovering the mouse over the square will reveal the index sequence.
    HINT! The tool can also suggest sequential indices while maintaining optimal nucleotide balance! Use the “Suggest Next Index” button to reveal the next ideal index to use in order (click also Help button for tips).
  5. OPTIONAL! Enter the relative molarity of each sample (by default molarity is set to 1 for each index).
  6. Check the graphic below the plate layout for a visualization of the relative nucleotide frequency (ACGT) at each position of the index read and overall laser color balance.
    HINT! The signal balance between red and green lasers is shown in a pie-chart format for each position of the index read. Ideally the signals should be balanced 50:50,whereas unbalanced signals should be avoided. The exact percentage is revealed if you hover over the pie charts.

Index Balance

It is important to ensure optimal color and nucleotide balance at each base of the index read to maintain base calling quality. In general, Illumina instruments two lasers (red and green) for excitation of fluorophores attached to the individual nucleotides. Depending on the type of chemistry, instruments have either 4-channel, or 2-channel imaging modes. The 4-channel instruments (HiSeq and MiSeq) use green lasers to detect G and T and red lasers to detect A and C. For the 2-channel instruments (MiniSeq, NextSeq, and NovaSeq), the green laser is used to detect T, and the red laser to detect C. A is detected by a mixture of red and green signal (appearing yellow), and the absence of any signal represents G.

Single or Dual Indexing?

Single-indexed libraries include only i7 indices, while dual-indexed, libraries are barcoded with both i7 and i5 indices. It is necessary to check the index read balance for the i7 and i5 index reads separately. The i7 indices are always sequenced in forward orientation. The i5 indices are sequenced either in forward (i5) or reverse-complement (i5rc) orientation. Information about i5 sequencing workflows can be found in the i5 Dual Indexing Add-on Kits Instruction Manual (047IM109). The table below shows the instruments that use forward and reverse-complement i5 index orientation, respectively:

i5 – Forward Orientation i5rc – Reverse Complement Orientation
Instrument Flow Cell Type* Instrument Flow Cell Type*
MiSeq® PE MiniSeq™ PE
HiSeq® 2500 /2000 SR, PE NextSeq® 500/550 PE
HiSeq® 3000/4000 SR only HiSeq® 3000/4000 PE only
NovaSeq™ 6000 PE

*SR = single-read, PE = paired-end.

Tips for Low Sample Number Multiplexing:

We recommend where possible to multiplex a minimum of 8 libraries (i.e. 8 indices) per lane. To ensure optimal nucleotide balance the indices should ideally be used column-wise from the i7 and i5 index plates (i.e. in multiples of 8), or in numerical order as the number of libraries to multiplex increases. For smaller numbers of libraries (i.e. less than 8). we can also suggest the following when pooling only 4 samples per lane:

  • i7 indices for single indexing:
    • Prepare four equimolar index mixes: 7001-7002 for one sample, 7003-7004 for the second sample, 7005-7006 for the third sample, and 7007-7008 for the fourth sample.
    • Add 5 μl of the index mix to the PCR for the library amplification.
    • Alternatively, 7006, 7008, 7023, and 7096, are an example of a well-balanced set or 4 individual indices.
    • The set of 4 indices provided in QuantSeq and SENSE Trial Kits are also perfectly balanced for color and nucleotide content.
  • i5 indices for dual indexing
    • Use indices 5001 – 5004 as these contain perfect nucleotide balance at each position of the index read.

Please note that the i7 Indices included in the Small RNA-Seq Library Prep Kits (Cat. No. 052, 058) are referred to as SRi7001 – SRi 7096. However, the index sequences and plate layout are the same as those for the i7 Index Plate for QuantSeq/SENSE Library Prep Kits (and Cat. No. 044.96).

ATTENTION: The i7 and Sri7 index primers are not interchangeable! Sri7 primers must only be used for Small RNA-Seq library prep and cannot be used for QuantSeq/SENSE library amplification. Similarly, the i5 and i7 Index Plates for QuantSeq/SENSE cannot be used for amplification of libraries prepared with the Small RNA-Seq Library Prep Kit.