Journal
BMC GENOMICS
Volume 17, Issue -, Pages -Publisher
BMC
DOI: 10.1186/s12864-016-3217-x
Keywords
Illumina; Sequencing; Multiplexing; Sequencing errors
Funding
- Simons Foundation
- Targeted Grant in the Mathematical Modeling of Living Systems Award [342039]
- National Science Foundation [DEB 1457518]
- National Institute of Food and Agriculture, US Department of Agriculture, Hatch project [1006261]
- Division Of Environmental Biology
- Direct For Biological Sciences [1457518] Funding Source: National Science Foundation
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Background: 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. Results: 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. Conclusions: 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.
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