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Low rate of index hopping on the Illumina HiSeq X platform

Tom van der Valk, Francesco Vezzi, Mattias Ormestad, View ORCID ProfileLove Dalén, View ORCID ProfileKaterina Guschanski
doi: https://doi.org/10.1101/179028
Tom van der Valk
1Animal Ecology, Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, 752 36, Uppsala, Sweden
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Francesco Vezzi
2Science for Life Laboratory, Tomtebodavägen 23A, 17165 Solna, Sweden
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Mattias Ormestad
2Science for Life Laboratory, Tomtebodavägen 23A, 17165 Solna, Sweden
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Love Dalén
3Department of Bioinformatics and Genetics, Swedish Museum of Natural History, SE-10405 Stockholm, Sweden
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Katerina Guschanski
1Animal Ecology, Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, 752 36, Uppsala, Sweden
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Abstract

The high throughput capacities of the Illumina sequencing platforms and the possibility to label samples with unique identifiers has encouraged a wide use of sample multiplexing. However, this practice results in low rates of read misassignment (<1%) across samples sequenced on the same lane on all Illumina sequencing platforms that rely on the traditional bridge amplification. Alarmingly high rates of read misassignment of up to 10% were recently reported for the newest Illumina machines (HiSeq X and HiSeq 4000). This potentially calls into question previously generated and published results and may make future use of these platforms prohibitive for many applications in biology and medicine. In this study we rely on inline barcodes that are ligated to both ends of the DNA insert, to directly quantify the amount of index hopping in historical museum-preserved samples. As the barcodes become part of the sequencing read, they allow us to reliably infer the read origin even in the presence of index hopping. After sequencing the same pooled library of seven samples on three independent HiSeq X lanes and accounting for multiple possible sources of error, including barcode and index cross-contamination, we identified on average only 0.470% hopped reads. We conclude that index hopping happens on the newest generation of Illumina sequencing platforms, but results in a similar rate of read missagnment as reported for older Illumina machines. We nonetheless recommend using inline barcodes in multiplexing studies that rely on low-coverage data, require absolute certainty and/or aim to characterize rare variants.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted August 22, 2017.
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Low rate of index hopping on the Illumina HiSeq X platform
Tom van der Valk, Francesco Vezzi, Mattias Ormestad, Love Dalén, Katerina Guschanski
bioRxiv 179028; doi: https://doi.org/10.1101/179028
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Low rate of index hopping on the Illumina HiSeq X platform
Tom van der Valk, Francesco Vezzi, Mattias Ormestad, Love Dalén, Katerina Guschanski
bioRxiv 179028; doi: https://doi.org/10.1101/179028

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