WFRF:(Osman Omneya Ahmed)
Sökning: WFRF:(Osman Omneya Ahmed) > Microbial Community...
- Sinclair, LucasUppsala universitet,Limnologi,Science for Life Laboratory, SciLifeLab (författare)
Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons : Evaluating the Illumina Platform
- Artikel/kapitelEngelska2015
Förlag, utgivningsår, omfång ...
- 2015-02-03
- Public Library of Science (PLoS),2015
- electronicrdacarrier
Nummerbeteckningar
- LIBRIS-ID:oai:DiVA.org:uu-243557
- https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-243557URI
- https://doi.org/10.1371/journal.pone.0116955DOI
Kompletterande språkuppgifter
- Språk:engelska
- Sammanfattning på:engelska
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- SwePubSwePub
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Anmärkningar
- As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner – with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition.
Ämnesord och genrebeteckningar
- NATURVETENSKAP Annan naturvetenskap hsv//swe
- NATURAL SCIENCES Other Natural Sciences hsv//eng
Biuppslag (personer, institutioner, konferenser, titlar ...)
- Ahmed Osman, OmneyaUppsala universitet,Science for Life Laboratory, SciLifeLab,Limnologi(Swepub:uu)omnah486 (författare)
- Bertilsson, StefanUppsala universitet,Science for Life Laboratory, SciLifeLab,Limnologi(Swepub:uu)stber839 (författare)
- Eiler, AlexanderUppsala universitet,Science for Life Laboratory, SciLifeLab,Limnologi(Swepub:uu)aleil133 (författare)
- Uppsala universitetLimnologi (creator_code:org_t)
Sammanhörande titlar
- Ingår i:PLOS ONE: Public Library of Science (PLoS)10:21932-6203
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