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PacBio library preparation using blunt-end adapter ligation produces significant artefactual fusion DNA sequences

Paul M Griffith, Castle Raley, David Sun, Yongmei Zhao, Zhonghe Sun, Monika Mehta, Bao Tran, Xiaolin Wu
doi: https://doi.org/10.1101/245241
Paul M Griffith
Leidos Biomedical Research Inc.
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Castle Raley
Leidos Biomedical Research Inc.
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David Sun
Leidos Biomedical Research Inc.
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Yongmei Zhao
Leidos Biomedical Research Inc.
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Zhonghe Sun
Leidos Biomedical Research Inc.
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Monika Mehta
Leidos Biomedical Research Inc.
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Bao Tran
Leidos Biomedical Research Inc.
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Xiaolin Wu
Leidos Biomedical Research Inc.
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  • For correspondence: forestwu@mail.nih.gov
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Abstract

Pacific Biosciences (PacBio) RS II sequencer, utilizing Single-Molecule, Real-Time (SMRT) technology, has revolutionized next-generation sequencing by providing an accurate long-read platform. PacBio single-molecule long reads have been used to delineate complex spliceoforms, detect mutations in highly homologous sequences, identify mRNA chimeras and chromosomal translocations, accurately haplotype phasing over multiple kilobase distances and aid in assembly of genomes with complex structural variation. The PacBio protocol for preparation of sequencing templates employs blunt-end hairpin adapter ligation, which enables a short turnaround time for sequence production. However, we have found a significant portion of sequencing yield contains chimeric reads resulting from blunt-end ligation of multiple template molecules to each other prior to adapter ligation. These artefactual fusion DNA sequences pose a major challenge to analysis and can lead to false-positive detection of fusion events. We assessed the frequency of artefactual fusion when using blunt-end adapter ligation and compared it to an alternative method using A/T overhang adapter ligation. The A/T overhang adapter ligation method showed a vast improvement in limiting artefactual fusion events and is now our recommended procedure for adapter ligation during PacBio library preparation.

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The copyright holder for this preprint is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license.
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  • Posted January 9, 2018.

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PacBio library preparation using blunt-end adapter ligation produces significant artefactual fusion DNA sequences
Paul M Griffith, Castle Raley, David Sun, Yongmei Zhao, Zhonghe Sun, Monika Mehta, Bao Tran, Xiaolin Wu
bioRxiv 245241; doi: https://doi.org/10.1101/245241
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PacBio library preparation using blunt-end adapter ligation produces significant artefactual fusion DNA sequences
Paul M Griffith, Castle Raley, David Sun, Yongmei Zhao, Zhonghe Sun, Monika Mehta, Bao Tran, Xiaolin Wu
bioRxiv 245241; doi: https://doi.org/10.1101/245241

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