Index Balance Checker
Lexogen Indices for Illumina (QuantSeq/SENSE Kits)
The Index Balance Checker tool will help you select the optimal index combination for multiplexed sequencing. Specifically, the applet evaluates the color balance of your chosen Lexogen i7 Indices (i7 Index Plate) and can be used on any number of samples for any Illumina machine (HiSeq, NextSeq, MiSeq, MiniSeq).
In general, Illumina uses two imaging modes on its next generation sequencers. Thereby, it is important to maintain color balance at each base of the index read, as otherwise index read sequencing could fail. In the 4-channel mode (HiSeq and MiSeq), green lasers are used to sequence G/T and red lasers to sequence A/C. In the 2-channel mode (NextSeq and MiniSeq), green laser is used to sequence A/T, red laser to sequence C, and absence of any signal represents G.
To determine the optimal combination of indices, start with selecting the kit size (8 preps, 24 preps, or 96 preps for SENSE kits and 24 preps or 96 preps for QuantSeq kits) and the Illumina machine you want to use for sequencing. After selecting the indices and adding the molarity of your samples, you will get a visualisation of the relative frequency of the nucleotides (ACGT) at each index position (1-6). If further barcodes are needed, the tool can suggest additional indices while keeping a good nucleotide balance. For a detailed explanation about the use of the Index Balance Checker, please read the
In general, we recommend processing a minimum of 8 indices per lane. If less than 8 samples are sequenced, use more than one index per sample in equimolar concentrations. Examples for 2 or 4 samples per lane are given below.
Two samples per lane: In the PCR use 5 μl of an equimolar mix of 7001-7004 for one sample and 5 μl of an equimolar mix of 7005-7008 for the second. Alternatively, two indices can be applied per sample but check the color balance using the evaluation. For instance, use 2.5 μl of 7006 and 2.5 μl of 7008 for one sample and 2.5 μl of 7023 and 2.5 μl of 7096 for the second. Here two indices are applied to each sample in order to balance the red and green laser signals.
Four samples per lane: In the PCR use 5 μl of an equimolar mix of 7001-7002 for one sample, 5 μl of an equimolar mix of 7003-7004 for the second, 5 μl of an equimolar mix of 7005-7006 for the third, and 5 μl of an equimolar mix of 7007-7008 for the fourth. Alternatively, when using only 1 index per sample we recommend checking the color balance with the evaluation tool. Indices 7006, 7008, 7023, and 7096 are examples of four well-balanced indices.