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Index Multiplex Compatibility Checker.

Check whether your NGS index/barcode pool is compatible for multiplexed sequencing. Evaluate per-cycle color balance, Hamming distance matrix, and minimum diversity for 4-channel, 2-channel, and 1-channel Illumina chemistries. Paste i5/i7 sequences or upload CSV. All computation runs client-side.

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Validated2026-04-07
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Load example Index Multiplex Compatibility Checker data to see the full workflow

Index Sequences

Paste index sequences, one per line. Formats accepted: name,sequence, name sequence, or just sequences (auto-named). You can also include a third column for read type (i5/i7).

Sequencing Settings

MiSeq, HiSeq, NovaSeq 6000

Applied when no read column is specified

When to use

  • Check if your chosen NGS indexes can be safely pooled for multiplexed sequencing
  • Evaluate color balance per cycle for your specific sequencing platform chemistry
  • Verify Hamming distance between all index pairs before committing to a sequencing run
  • Get swap suggestions when your pool has color-balance or distance problems

Do not use for

  • Designing new index sequences from scratch — use your vendor’s index design tool
  • Checking dual-indexed (i5 + i7) combinatorial compatibility — this tool checks each index read independently
  • Library preparation or pooling calculations — use a molarity calculator instead

Always check both i5 and i7 reads separately

If your library uses dual indexing, run this tool once for i7 indexes and once for i5 indexes. Both reads must independently have good color balance and sufficient Hamming distance.

Add PhiX spike-in for small pools

Pools with fewer than 4 unique indexes are at high risk for color-balance problems. A 10–20% PhiX spike-in provides the base diversity the sequencer needs for accurate cluster identification and phasing estimation.

Unique Dual Indexes (UDI) prevent index hopping

On patterned flow cells (NovaSeq, NextSeq 2000), index hopping can misassign up to 0.1–2% of reads. UDI pairs eliminate this by requiring both i5 and i7 to match, making hopping detectable.

Check your platform’s chemistry before choosing indexes

2-channel platforms (NextSeq 1000/2000, NovaSeq X) have stricter color-balance requirements than 4-channel platforms. An index pool that works on MiSeq may fail on NextSeq.

Beware of 6-bp vs 8-bp index mixups

Mixing 6-bp and 8-bp indexes in the same pool causes demultiplexing failures. Always confirm that all indexes in your pool have the same length and match the sequencer’s index read configuration.

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Method

The tool calculates pairwise Hamming distances between all index sequences and checks that the minimum is ≥\geq 3 (allowing 1-mismatch demultiplexing). Per-cycle color balance is evaluated according to the selected chemistry: 4-channel requires all 4 bases; 2-channel requires signal in both green (A/T) and red (A/C) channels; 1-channel requires diversity across both imaging steps. A minimum of 3 distinct bases per cycle is recommended. Swap suggestions are generated when over-represented bases are detected.

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Validated

Last validated 2026-04-07. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.

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How to cite

How to Cite

ConductScience NGS Index Multiplex Compatibility Checker (v1.23.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/index-multiplex-compatibility-checker

Illumina, Inc. Index Adapter Pooling Guide. Illumina Technical Note. 2023. Document #1000000041074. doi:1000000041074

Krueger F et al. Large Scale Loss of Data in Low-Diversity Illumina Sequencing Libraries Can Be Recovered by Deferred Cluster Calling. PLoS ONE. 2011;6:e16607. doi:10.1371/journal.pone.0016607

Costello M et al. Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms. BMC Genomics. 2018;19:332. doi:10.1186/s12864-018-4703-0

How NGS index design works

NGS indexes (barcodes) are short synthetic DNA sequences ligated to each sample library before pooling. During sequencing, a separate "index read" decodes each fragment’s sample origin.

Key design principles:

1. Sufficient Hamming distance: Each pair of indexes in a pool should differ by ≥\geq 3 positions, allowing error correction during demultiplexing.

2. Color balance: Every cycle of the index read should have adequate representation of all four bases (or at minimum, signal in both fluorescent channels) for the sequencer to calibrate correctly.

3. No homopolymer runs: Long runs of the same base reduce sequencing quality.

4. Balanced GC content: Extreme GC bias in indexes can cause secondary structures or biased amplification.

Understanding color balance by chemistry type

4-channel SBS (MiSeq, HiSeq): Each of the four bases emits a distinct fluorescent color. The sequencer needs all four signals for accurate base calling. If only one or two bases are present at a cycle, phasing/pre-phasing estimation fails.
2-channel SBS (NextSeq, NovaSeq X): Two dye channels (green and red). A = both channels, C = red only, T = green only, G = dark (no signal). The sequencer needs signal in both channels: at least one A or T (for green) and at least one A or C (for red). A pool of only G and T would leave the red channel empty.
1-channel (iSeq 100): Two sequential images. Image 1: A+T lit. Image 2: A+C lit. G = dark in both. Similar balance requirements as 2-channel but with imaging-based detection.

Demultiplexing and error correction

After sequencing, bcl2fastq or DRAGEN demultiplexes reads by matching index reads to the expected index sequences.

  • 0 mismatches: Strictest setting. Only exact matches are assigned. Highest confidence but loses reads with sequencing errors in the index.
  • 1 mismatch (default): Allows one base difference. Requires Hamming distance ≥\geq 3 between all index pairs to ensure unambiguous assignment.
  • 2 mismatches: Allows two differences. Requires Hamming distance ≥\geq 5. Rarely used.

Reads that cannot be unambiguously assigned are placed in the "Undetermined" bin. High undetermined rates (> 5–10%) suggest index problems.

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