Show Notes
White R et al., F1000Research - Nanopore MinION sequencing of murine Ifna, Ifnb and Actb amplicons identified post-amplification chimeric reads during library ligation, with ~1.7% of mapped reads containing chimeric elements.
Study Highlights: cDNA from a Nippostrongylus brasiliensis–treated mouse amplified for Ifna family, Ifnb and Actb amplicons.
Key methods: separate PCR amplicons were barcoded, ligated using two methods (quick vs overnight), sequenced on an Oxford Nanopore MinION and base-called/mapped with Albacore and LAST. Main quantitative result: 4,563 reads (≈1.7% of amplicon/barcode-mappable 1D reads) were classified as definitively chimeric, with repeated identical amplicons most common and overnight ligation associated with more repeated-amplicon chimeras than quick ligation.
Functional implication: post-amplification ligation during library prep can create detectable chimeric reads that long-read nanopore data and raw-signal inspection can identify and allow filtering, with implications for amplicon sequencing workflows and index switching concerns.
Conclusion: Post-amplification ligation during library preparation produced detectable chimeric reads (~1.7%), and including identifiable adapters with careful long-read QC enables detection and exclusion of most chimeras.
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Reference:
White R, Pellefigues C, Ronchese F, Lamiable O, Eccles D. Investigation of chimeric reads using the MinION [version 2; peer review: 2 approved]. F1000Research. 2017;6:631. https://doi.org/10.12688/f1000research.11547.2
License:
This episode is based on an open-access article published under the Creative Commons Attribution 4.0 International License (CC BY 4.0) - https://creativecommons.org/licenses/by/4.0/
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