A primer-independent DNA polymerase-based method for competent whole-genome amplification of intermediate to high GC sequences
Entity
UAM. Departamento de BioquímicaPublisher
Oxford University PressDate
2023-08-21Citation
10.1093/nargab/lqad073
NAR Genomics and Bioinformatics 5.3 (2023): lqad073
ISSN
2631-9268DOI
10.1093/nargab/lqad073Funded by
MCIN/AEI/10.13039/501100011033 and ERDF A way of making Europe [PGC2018-09723-A-I00 and PID2021-123403NB-I00 to M.R.R.]; C. Egas’ laboratory was funded by the European Union′s Horizon 2020 Research and Innovation Program [685474]; METAFLUIDICS project; C.D.O. and C.M.C. were holder of Fellowships from the Spanish Ministry of University [FPU16/02665] and Spanish Ministry of Science and Innovation [PRE2019-087304], respectivelyProject
info:eu-repo/grantAgreement/AEI//PGC2018-09723-A-I00/ES/DNA POLIMERASAS INDEPENDIENTES DE PRIMER Y SUS APLICACIONES EN BIOTECNOLOGIA Y BIOMEDICINA/; info:eu-repo/grantAgreement/AEI//PID2021-123403NB-I00; info:eu-repo/grantAgreement/EC/H2020/685474/EU//METAFLUIDICSEditor's Version
https://doi.org/10.1093/nargab/lqad073Subjects
Multiple displacement amplification (MDA); DNA; MDA protocols; piPoIB; piMDA; metagenomics; Biología y Biomedicina / BiologíaNote
The dataset (raw sequencing data) that supports the findings of this study are archived in the Universidad Autónoma de Madrid data repository e‐cienciaDatos in DOI: 10.21950/HCNDGRights
© The Author(s) 2023. Published by Oxford University Press on behalf of NAR Genomics and BioinformaticsAbstract
Multiple displacement amplification (MDA) has proven to be a useful technique for obtaining large amounts of DNA from tiny samples in genomics and metagenomics. However, MDA has limitations, such as amplification artifacts and biases that can interfere with subsequent quantitative analysis. To overcome these challenges, alternative methods and engineered DNA polymerase variants have been developed. Here, we present new MDA protocols based on the primer-independent DNA polymerase (piPolB), a replicative-like DNA polymerase endowed with DNA priming and proofreading capacities. These new methods were tested on a genomes mixture containing diverse sequences with high-GC content, followed by deep sequencing. Protocols relying on piPolB as a single enzyme cannot achieve competent amplification due to its limited processivity and the presence of ab initio DNA synthesis. However, an alternative method called piMDA, which combines piPolB with Φ29 DNA polymerase, allows proficient and faithful amplification of the genomes. In addition, the prior denaturation step commonly performed in MDA protocols is dispensable, resulting in a more straightforward protocol. In summary, piMDA outperforms commercial methods in the amplification of genomes and metagenomes containing high GC sequences and exhibits similar profiling, error rate and variant determination as the non-amplified samples
Files in this item
Google Scholar:Ordoñez, Carlos D.
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Mayoral Campos, Carmen
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Conceição, Egas
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Redrejo Rodríguez, Modesto
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Except where otherwise noted, this item's license is described as © The Author(s) 2023. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics
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