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BMC Genomics. 2016 Nov 4;17(1):876.

Quality filtering of Illumina index reads mitigates sample cross-talk.

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Department of Bacteriology, UW-Madison, Madison, USA.
Wisconsin Institute for Discovery, UW-Madison, 330 N. Orchard St, Madison, 53715, WI, USA.
Department of Bacteriology, UW-Madison, Madison, USA.
Wisconsin Institute for Discovery, UW-Madison, 330 N. Orchard St, Madison, 53715, WI, USA.



Multiplexing multiple samples during Illumina sequencing is a common practice and is rapidly growing in importance as the throughput of the platform increases. Misassignments during de-multiplexing, where sequences are associated with the wrong sample, are an overlooked error mode on the Illumina sequencing platform. This results in a low rate of cross-talk among multiplexed samples and can cause detrimental effects in studies requiring the detection of rare variants or when multiplexing a large number of samples.


We observed rates of cross-talk averaging 0.24 % when multiplexing 14 different samples with unique i5 and i7 index sequences. This cross-talk rate corresponded to 254,632 misassigned reads on a single lane of the Illumina HiSeq 2500. Notably, all types of misassignment occur at similar rates: incorrect i5, incorrect i7, and incorrect sequence reads. We demonstrate that misassignments can be nearly eliminated by quality filtering of index reads while preserving about 90 % of the original sequences.


Cross-talk among multiplexed samples is a significant error mode on the Illumina platform, especially if samples are only separated by a single unique index. Quality filtering of index sequences offers an effective solution to minimizing cross-talk among samples. Furthermore, we propose a straightforward method for verifying the extent of cross-talk between samples and optimizing quality score thresholds that does not require additional control samples and can even be performed post hoc on previous runs.


Illumina; Multiplexing; Sequencing; Sequencing errors

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