Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform
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{"title"=>"Microbial community composition and diversity via 16S rRNA gene amplicons: Evaluating the illumina platform", "type"=>"journal", "authors"=>[{"first_name"=>"Lucas", "last_name"=>"Sinclair", "scopus_author_id"=>"56675539100"}, {"first_name"=>"Omneya Ahmed", "last_name"=>"Osman", "scopus_author_id"=>"24778732200"}, {"first_name"=>"Stefan", "last_name"=>"Bertilsson", "scopus_author_id"=>"6603932886"}, {"first_name"=>"Alexander", "last_name"=>"Eiler", "scopus_author_id"=>"6506469534"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "arxiv"=>"1310.0424", "scopus"=>"2-s2.0-84922385648", "pui"=>"602005554", "doi"=>"10.1371/journal.pone.0116955", "isbn"=>"1932-6203", "sgr"=>"84922385648", "pmid"=>"25647581"}, "id"=>"0797ce16-f1b3-3ae3-9f74-c06a0b297b9f", "abstract"=>"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.", "link"=>"http://www.mendeley.com/research/microbial-community-composition-diversity-via-16s-rrna-gene-amplicons-evaluating-illumina-platform", "reader_count"=>443, "reader_count_by_academic_status"=>{"Unspecified"=>16, "Professor > Associate Professor"=>7, "Librarian"=>1, "Researcher"=>101, "Student > Doctoral Student"=>36, "Student > Ph. D. Student"=>137, "Student > Postgraduate"=>12, "Student > Master"=>77, "Other"=>15, "Student > Bachelor"=>27, "Lecturer"=>1, "Lecturer > Senior Lecturer"=>4, "Professor"=>9}, "reader_count_by_user_role"=>{"Unspecified"=>16, "Professor > Associate Professor"=>7, "Librarian"=>1, "Researcher"=>101, "Student > Doctoral Student"=>36, "Student > Ph. D. 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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1879303"], "description"=>"<p>The precision of the method over all pools and barcodes is evaluated. For beta-diversity measures, a permutational MANOVA test was used. For alpha-diversity measures, a permutational ANOVA test is used. The effect of methods, barcodes and the combination of methods and barcodes are given.</p><p>Precision of the Illumina method.</p>", "links"=>[], "tags"=>["Microbial Community Composition", "DNA quantification procedures", "16 S rRNA gene amplicons", "50 barcode pairs", "sequencing", "laboratories switch vendors", "pcr", "beta diversity estimates", "method", "UPARSE", "qiime", "MiSeq Illumina platform", "beta diversity", "sample", "Illumina sequence data", "otu"], "article_id"=>1300437, "categories"=>["Uncategorised"], "users"=>["Lucas Sinclair", "Omneya Ahmed Osman", "Stefan Bertilsson", "Alexander Eiler"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0116955.t001", "stats"=>{"downloads"=>2, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Precision_of_the_Illumina_method_/1300437", "title"=>"Precision of the Illumina method.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-02-03 02:57:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1879302"], "description"=>"<p>This dendrogram included 24 replicates of our test sediment sample sequenced on the Illumina platform as well as the same sample sequenced on a 454 machine. In addition, 9 soda lakes samples that were equally sequenced on both technologies are included. For the soda lakes, the letters correspond to different systems whereas the numbers correspond to different sampling dates. Using the UniFrac metric to compute a distance matrix, a hierarchical clustering is performed following the UPGMA method. The detail of the taxonomic composition can be seen in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116955#pone.0116955.s007\" target=\"_blank\">S7 Fig.</a></p>", "links"=>[], "tags"=>["Microbial Community Composition", "DNA quantification procedures", "16 S rRNA gene amplicons", "50 barcode pairs", "sequencing", "laboratories switch vendors", "pcr", "beta diversity estimates", "method", "UPARSE", "qiime", "MiSeq Illumina platform", "beta diversity", "sample", "Illumina sequence data", "otu"], "article_id"=>1300436, "categories"=>["Uncategorised"], "users"=>["Lucas Sinclair", "Omneya Ahmed Osman", "Stefan Bertilsson", "Alexander Eiler"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0116955.g004", "stats"=>{"downloads"=>0, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Correspondence_of_phylogenetic_distance_between_454_and_Illumina_/1300436", "title"=>"Correspondence of phylogenetic distance between 454 and Illumina.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-03 02:57:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1879299"], "description"=>"<p>To assign an Illumina read to a particular sample, one examines both of the barcodes at each end of the sequence. In green, the two barcodes agree on which sample the read is coming from. In black, no barcodes are found on either end. In yellow, only one barcode is present. In orange, the two barcodes come from the same directional set and should not be found together (e.g. two forward barcodes). In red, the two barcodes each indicate a different sample. Overall, with this setup, about half of the raw data is discarded.</p>", "links"=>[], "tags"=>["Microbial Community Composition", "DNA quantification procedures", "16 S rRNA gene amplicons", "50 barcode pairs", "sequencing", "laboratories switch vendors", "pcr", "beta diversity estimates", "method", "UPARSE", "qiime", "MiSeq Illumina platform", "beta diversity", "sample", "Illumina sequence data", "otu"], "article_id"=>1300433, "categories"=>["Uncategorised"], "users"=>["Lucas Sinclair", "Omneya Ahmed Osman", "Stefan Bertilsson", "Alexander Eiler"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0116955.g001", "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Distribution_of_barcodes_matching_and_mismatching_/1300433", "title"=>"Distribution of barcodes matching and mismatching.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-03 02:57:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1879309", "https://ndownloader.figshare.com/files/1879310", "https://ndownloader.figshare.com/files/1879311", "https://ndownloader.figshare.com/files/1879312", "https://ndownloader.figshare.com/files/1879313", "https://ndownloader.figshare.com/files/1879314", "https://ndownloader.figshare.com/files/1879315", "https://ndownloader.figshare.com/files/1879316", "https://ndownloader.figshare.com/files/1879317", "https://ndownloader.figshare.com/files/1879318", "https://ndownloader.figshare.com/files/1879319", "https://ndownloader.figshare.com/files/1879320", "https://ndownloader.figshare.com/files/1879321"], "description"=>"<div><p>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.</p></div>", "links"=>[], "tags"=>["Microbial Community Composition", "DNA quantification procedures", "16 S rRNA gene amplicons", "50 barcode pairs", "sequencing", "laboratories switch vendors", "pcr", "beta diversity estimates", "method", "UPARSE", "qiime", "MiSeq Illumina platform", "beta diversity", "sample", "Illumina sequence data", "otu"], "article_id"=>1300441, "categories"=>["Uncategorised"], "users"=>["Lucas Sinclair", "Omneya Ahmed Osman", "Stefan Bertilsson", "Alexander Eiler"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0116955.s001", "https://dx.doi.org/10.1371/journal.pone.0116955.s002", "https://dx.doi.org/10.1371/journal.pone.0116955.s003", "https://dx.doi.org/10.1371/journal.pone.0116955.s004", "https://dx.doi.org/10.1371/journal.pone.0116955.s005", "https://dx.doi.org/10.1371/journal.pone.0116955.s006", "https://dx.doi.org/10.1371/journal.pone.0116955.s007", "https://dx.doi.org/10.1371/journal.pone.0116955.s008", "https://dx.doi.org/10.1371/journal.pone.0116955.s009", "https://dx.doi.org/10.1371/journal.pone.0116955.s010", "https://dx.doi.org/10.1371/journal.pone.0116955.s011", "https://dx.doi.org/10.1371/journal.pone.0116955.s012", "https://dx.doi.org/10.1371/journal.pone.0116955.s013"], "stats"=>{"downloads"=>23, "page_views"=>49, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Microbial_Community_Composition_and_Diversity_via_16S_rRNA_Gene_Amplicons_Evaluating_the_Illumina_Platform_/1300441", "title"=>"Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-02-03 02:57:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1879304"], "description"=>"<p>A linear model was fitted to the alpha diversity estimates from 454 and Illumina data for each sample. Regressions are plotted in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116955#pone.0116955.s010\" target=\"_blank\">S10 Fig.</a></p><p>Correspondence between 454 and Illumina.</p>", "links"=>[], "tags"=>["Microbial Community Composition", "DNA quantification procedures", "16 S rRNA gene amplicons", "50 barcode pairs", "sequencing", "laboratories switch vendors", "pcr", "beta diversity estimates", "method", "UPARSE", "qiime", "MiSeq Illumina platform", "beta diversity", "sample", "Illumina sequence data", "otu"], "article_id"=>1300438, "categories"=>["Uncategorised"], "users"=>["Lucas Sinclair", "Omneya Ahmed Osman", "Stefan Bertilsson", "Alexander Eiler"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0116955.t002", "stats"=>{"downloads"=>2, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Correspondence_between_454_and_Illumina_/1300438", "title"=>"Correspondence between 454 and Illumina.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2015-02-03 02:57:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1879301"], "description"=>"<p>Every one of our sediment sample replicates, i.e. the triplicate two-step PCR, the one-step PCR and the updated chemistry are separated into three size fractions (low, medium, high) according to the peaks identified in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116955#pone.0116955.g002\" target=\"_blank\">Fig. 2</a>. Fragments below 446 bp are exclusively originating from archaea. The second peak between 447 and 464 bp contains, for instance, contains a majority of the Chloroflexi. The last group above 465 bp holds most of the Bacteroidetes. Other species such as Proteobacteria or Firmicutes are found spanning a size range in the environmental sample.</p>", "links"=>[], "tags"=>["Microbial Community Composition", "DNA quantification procedures", "16 S rRNA gene amplicons", "50 barcode pairs", "sequencing", "laboratories switch vendors", "pcr", "beta diversity estimates", "method", "UPARSE", "qiime", "MiSeq Illumina platform", "beta diversity", "sample", "Illumina sequence data", "otu"], "article_id"=>1300435, "categories"=>["Uncategorised"], "users"=>["Lucas Sinclair", "Omneya Ahmed Osman", "Stefan Bertilsson", "Alexander Eiler"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0116955.g003", "stats"=>{"downloads"=>2, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Composition_of_different_lengths_fractions_/1300435", "title"=>"Composition of different lengths fractions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-03 02:57:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/1879300"], "description"=>"<p>Once all the forward and reverse reads from the Illumina sequencer are joined one can see – in gray here – a pattern in the size of the fragments produced. Superposing in red the abundance of length variation found in the SILVAMOD database, one can see that the variation we uncovered follows closely the natural variation of the V3–V4 region of the 16S rRNA gene. As shown in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116955#pone.0116955.g003\" target=\"_blank\">Fig. 3</a>, each of the three peaks are composed of characteristic phyla.</p>", "links"=>[], "tags"=>["Microbial Community Composition", "DNA quantification procedures", "16 S rRNA gene amplicons", "50 barcode pairs", "sequencing", "laboratories switch vendors", "pcr", "beta diversity estimates", "method", "UPARSE", "qiime", "MiSeq Illumina platform", "beta diversity", "sample", "Illumina sequence data", "otu"], "article_id"=>1300434, "categories"=>["Uncategorised"], "users"=>["Lucas Sinclair", "Omneya Ahmed Osman", "Stefan Bertilsson", "Alexander Eiler"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0116955.g002", "stats"=>{"downloads"=>1, "page_views"=>26, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Distribution_of_sequence_lengths_for_matching_barcodes_/1300434", "title"=>"Distribution of sequence lengths for matching barcodes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-02-03 02:57:09"}

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{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}
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