Optimization of Protocol for Construction of Fungal ITS Amplicon Library for High-Throughput Illumina Sequencing to Study the Mycobiome of Aspen Leaves

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dc.authoridSiddique, Abu Bakar/0000-0001-5374-2348
dc.authorid, Abu Bakar Siddique/0000-0002-3178-523X
dc.authorscopusid57159118100
dc.authorscopusid6603337389
dc.authorscopusid15069243700
dc.authorscopusid57210685703
dc.authorwosidSiddique, Abu Bakar/ABW-4686-2022
dc.authorwosid, Abu Bakar Siddique/R-1776-2018
dc.contributor.authorAlbrectsen, Benedicte Riber
dc.contributor.authorAlbrectsen, Benedicte Riber
dc.contributor.authorIlbi, Hulya
dc.contributor.authorSiddique, Abu Bakar
dc.date.accessioned2023-01-12T20:15:28Z
dc.date.available2023-01-12T20:15:28Z
dc.date.issued2022
dc.departmentN/A/Departmenten_US
dc.description.abstractHigh-Throughput Illumina Sequencing (HTS) can be used to study metagenomes, for example, those of importance for plant health. However, protocols must be optimized according to the plant system in question, the focal microorganisms in the samples, the marker genes selected, and the number of environmental samples. We optimized the protocol for metagenomic studies of aspen leaves, originating from varied genotypes sampled across the growing season, and consequently varying in phenolic composition and in the abundance of endo- and epiphytic fungal species. We optimized the DNA extraction protocol by comparing commercial kits and evaluating five fungal ribosomal specific primers (Ps) alone, and with extended primers that allow binding to sample-specific index primers, and we then optimized the amplification with these composite Ps for 380 samples. The fungal DNA concentration in the samples varied from 561 ng/mu L to 1526 ng/mu L depending on the DNA extraction kit used. However, binding to phenolic compounds affected DNA quality as assessed by Nanodrop measurements (0.63-2.04 and 0.26-2.00 absorbance ratios for 260/280 and 260/230, respectively), and this was judged to be more important in making our choice of DNA extraction kit. We initially modified the PCR conditions after determining the concentration of DNA extract in a few subsamples and then evaluated and optimized the annealing temperature, duration, and number of cycles to obtain the required amplification and PCR product bands. For three specific Ps, the extended Ps produced dimers and unexpected amplicon fragments due to nonspecific binding. However, we found that the specific Ps that targeted the ITS2 region of fungal rDNA successfully amplified this region for every sample (with and without the extension PP) resulting in the desired PCR bands, and also allowing the addition of sample-specific index primers, findings which were successfully verified in a second PCR. The optimized protocol allowed us to successfully prepare an amplicon library in order to subject the intended 380 environmental samples to HTS.en_US
dc.description.sponsorshipKnut and Alice Wallenberg Foundation; Swedish Governmental Agency for Innovation Systems (VINNOVA) [KAW 2016.0341, KAW 2016.0352]; UPSCb; BRA by the UPSC Berzelii Centre for Forest Biotechnology; Trees for the Future (T4F) project; [586618-EPP-1-2017-1-FR-EPPKA1-JMD-MOB]; [JCK-1919.1]; [VINNOVA 2016-00504]en_US
dc.description.sponsorshipFinancial contributions from the Knut and Alice Wallenberg Foundation and the Swedish Governmental Agency for Innovation Systems (VINNOVA; reference numbers: VINNOVA 2016-00504; KAW 2016.0341 and KAW 2016.0352) to UPSC and UPSCb supported the study. Funding was provided by BRA supported by the UPSC Berzelii Centre for Forest Biotechnology and the Trees for the Future (T4F) project.en_US
dc.identifier.doi10.3390/app12031136
dc.identifier.issn2076-3417
dc.identifier.issn2076-3417en_US
dc.identifier.issue3en_US
dc.identifier.scopus2-s2.0-85123059952en_US
dc.identifier.scopusqualityQ2en_US
dc.identifier.urihttps://doi.org/10.3390/app12031136
dc.identifier.urihttps://hdl.handle.net/11454/78486
dc.identifier.volume12en_US
dc.identifier.wosWOS:000760258200001en_US
dc.identifier.wosqualityQ2en_US
dc.indekslendigikaynakWeb of Scienceen_US
dc.indekslendigikaynakScopusen_US
dc.language.isoenen_US
dc.publisherMdpien_US
dc.relation.ispartofApplied Sciences-Baselen_US
dc.relation.publicationcategoryMakale - Uluslararası Hakemli Dergi - Kurum Öğretim Elemanıen_US
dc.rightsinfo:eu-repo/semantics/openAccessen_US
dc.subjectNGSen_US
dc.subjectmetabarcodingen_US
dc.subjectmetagenomicsen_US
dc.subjecteDNAen_US
dc.subjectITSen_US
dc.subjectendophytesen_US
dc.subjectampliconen_US
dc.subjectaspenen_US
dc.subjectrDNAen_US
dc.subjectCommunity Analysisen_US
dc.subjectNeedle Mycobiomeen_US
dc.subjectPicea-Glaucaen_US
dc.subjectSystemen_US
dc.subjectEliminationen_US
dc.subjectAssemblagesen_US
dc.subjectDiversityen_US
dc.subjectElevationen_US
dc.subjectRecoveryen_US
dc.subjectPcren_US
dc.titleOptimization of Protocol for Construction of Fungal ITS Amplicon Library for High-Throughput Illumina Sequencing to Study the Mycobiome of Aspen Leavesen_US
dc.typeArticleen_US

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