Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi.

التفاصيل البيبلوغرافية
العنوان:	Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi.
المؤلفون:	U'Ren JM; School of Plant Sciences, University of Arizona, Tucson, AZ, 85721, USA., Riddle JM, Monacell JT, Carbone I, Miadlikowska J, Arnold AE
المصدر:	Molecular ecology resources [Mol Ecol Resour] 2014 Sep; Vol. 14 (5), pp. 1032-48. Date of Electronic Publication: 2014 Apr 10.
نوع المنشور:	Comparative Study; Evaluation Study; Journal Article; Research Support, U.S. Gov't, Non-P.H.S.
اللغة:	English
بيانات الدورية:	Publisher: Blackwell Country of Publication: England NLM ID: 101465604 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1755-0998 (Electronic) Linking ISSN: 1755098X NLM ISO Abbreviation: Mol Ecol Resour Subsets: MEDLINE
أسماء مطبوعة:	Original Publication: Oxford, England : Blackwell
مواضيع طبية MeSH:	Biota* , Diagnostic Errors, DNA Primers/genetics , Endophytes/classification , Fungi/classification , Preservation, Biological/methods , Specimen Handling/methods, Endophytes/genetics ; Endophytes/isolation & purification ; Fungi/genetics ; Fungi/isolation & purification ; Molecular Sequence Data ; Plants/microbiology ; Sequence Analysis, DNA/methods
مستخلص:	Next-generation sequencing technologies have provided unprecedented insights into fungal diversity and ecology. However, intrinsic biases and insufficient quality control in next-generation methods can lead to difficult-to-detect errors in estimating fungal community richness, distributions and composition. The aim of this study was to examine how tissue storage prior to DNA extraction, primer design and various quality-control approaches commonly used in 454 amplicon pyrosequencing might influence ecological inferences in studies of endophytic and endolichenic fungi. We first contrast 454 data sets generated contemporaneously from subsets of the same plant and lichen tissues that were stored in CTAB buffer, dried in silica gel or freshly frozen prior to DNA extraction. We show that storage in silica gel markedly limits the recovery of sequence data and yields a small fraction of the diversity observed by the other two methods. Using lichen mycobiont sequences as internal positive controls, we next show that despite careful filtering of raw reads and utilization of current best-practice OTU clustering methods, homopolymer errors in sequences representing rare taxa artificially increased estimates of richness c. 15-fold in a model data set. Third, we show that inferences regarding endolichenic diversity can be improved using a novel primer that reduces amplification of the mycobiont. Together, our results provide a rationale for selecting tissue treatment regimes prior to DNA extraction, demonstrate the efficacy of reducing mycobiont amplification in studies of the fungal microbiomes of lichen thalli and highlight the difficulties in differentiating true information about fungal biodiversity from methodological artefacts. (© 2014 John Wiley & Sons Ltd.)
فهرسة مساهمة:	Keywords: 454 amplicon pyrosequencing; ITSrDNA; Peltigera; Pinus; diversity; lichen; moss; mycobiont
سلسلة جزيئية:	GENBANK KJ508220; KJ508221; KJ508222; KJ508223; KJ508224; KJ508225; KJ508226; KJ508227; KJ508228; KJ508229; KJ508230; KJ508231; KJ508232; KJ508233; KJ508234; KJ508235; KJ508236; KJ508237; KJ508238; KJ508239; KJ508240; KJ508241; KJ508242; KJ508243; KJ508244; KJ508245; KJ508246; KJ508247; KJ508248; KJ508249; KJ508250; KJ508251; KJ508252; KJ508253; KJ508254; KJ508255; KJ508256; KJ508257; KJ508258; KJ508259; KJ508260; KJ508261; KJ508262; KJ508263; KJ508264; KJ508265; KJ508266; KJ508267; KJ508268; KJ508269; KJ508270; KJ508271; KJ508272; KJ508273; KJ508274; KJ508275; KJ508276; KJ508277; KJ508278; KJ508279; KJ508280; KJ508281; KJ508282; KJ508283; KJ508284; KJ508285; KJ508286; KJ508287; KJ508288; KJ508289; KJ508290; KJ508291; KJ508292; KJ508293; KJ508294; KJ508295; KJ508296; KJ508297; KJ508298; KJ508299; KJ508300; KJ508301; KJ508302; KJ508303; KJ508304; KJ508305; KJ508306; KJ508307; KJ508308; KJ508309; KJ508310; KJ508311; KJ508312; KJ508313; KJ508314; KJ508315; KJ508316; KJ508317; KJ508318; KJ508319; KJ508320; KJ508321; KJ508322; KJ508323; KJ508324; KJ508325; KJ508326; KJ508327; KJ508328; KJ508329; KJ508330; KJ508331; KJ508332; KJ508333; KJ508334; KJ508335; KJ508336; KJ508337; KJ508338; KJ508339; KJ508340; KJ508341; KJ508342
المشرفين على المادة:	0 (DNA Primers)
تواريخ الأحداث:	Date Created: 20140318 Date Completed: 20150401 Latest Revision: 20191210
رمز التحديث:	20221213
DOI:	10.1111/1755-0998.12252
PMID:	24628864
قاعدة البيانات:	MEDLINE

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تدمد:	1755-0998
DOI:	10.1111/1755-0998.12252