59 research outputs found

    Can Abundance of Protists Be Inferred from Sequence Data: A Case Study of Foraminifera

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    <div><p>Protists are key players in microbial communities, yet our understanding of their role in ecosystem functioning is seriously impeded by difficulties in identification of protistan species and their quantification. Current microscopy-based methods used for determining the abundance of protists are tedious and often show a low taxonomic resolution. Recent development of next-generation sequencing technologies offered a very powerful tool for studying the richness of protistan communities. Still, the relationship between abundance of species and number of sequences remains subjected to various technical and biological biases. Here, we test the impact of some of these biological biases on sequence abundance of SSU rRNA gene in foraminifera. First, we quantified the rDNA copy number and rRNA expression level of three species of foraminifera by qPCR. Then, we prepared five mock communities with these species, two in equal proportions and three with one species ten times more abundant. The libraries of rDNA and cDNA of the mock communities were constructed, Sanger sequenced and the sequence abundance was calculated. The initial species proportions were compared to the raw sequence proportions as well as to the sequence abundance normalized by rDNA copy number and rRNA expression level per species. Our results showed that without normalization, all sequence data differed significantly from the initial proportions. After normalization, the congruence between the number of sequences and number of specimens was much better. We conclude that without normalization, species abundance determination based on sequence data was not possible because of the effect of biological biases. Nevertheless, by taking into account the variation of rDNA copy number and rRNA expression level we were able to infer species abundance, suggesting that our approach can be successful in controlled conditions.</p> </div

    Proportions of cDNA sequences of <i>Allogromia</i>, <i>Rosalina</i>, and <i>Bolivina</i> found in the five mock communities.

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    <p>(A) Observed proportions of cDNA sequences without normalization; (B) Observed proportions of cDNA sequences after normalization with the ELF (Expression Level Factor, see text for details). Raw and normalized proportions were used for Chi<sup>2</sup> goodness of fit test (df = 2; α = 0.05). ***: significant at 0.001; **: significant at 0.01; *: significant at 0.05; arrow: not significant, meaning that observed proportions did not differ significantly from expected proportions. Mix 3 - with three cells of each species; mix 10 - with ten cells of each species; mix Allogromia – with thirty cells of <i>Allogromia</i> and three cells of <i>Rosalina</i> and <i>Bolivina</i>; mix Rosalina – with thirty cells of <i>Rosalina</i> and three cells of <i>Allogromia</i> and <i>Bolivina</i>; mix Bolivina – with thirty cells of <i>Bolivina</i> and three cells of <i>Allogromia</i> and <i>Rosalina</i>.</p

    Results of qPCR assays.

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    <p>(A) SSU rDNA copy number estimation per cell per species inferred from qPCR data, based on two replicate measurements. (B) SSU rRNA expression level per cell per species inferred from qPCR data based on one replicate measurement. Error bars are standard deviation.</p

    Elphidiids complete SSU rDNA phylogeny.

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    <p>Bayesian phylogeny implemented using the GTR+<i>Γ</i> model of evolution. RAxML bootstrap values and MrBayes posterior probabilities are shown at the nodes and solid circles indicate maximum node support (100/1.0). For each sequence, the DNA number is followed by the sampling location: CA (Halifax), RU (White Sea), NL (North Sea), CH (Patagonia), MED (Mediterranean Sea) and BRET (Brittany) and by the GenBank accession number.</p

    R and P values (Pearson test) of the relationships between the OTU proportions obtained with Sanger-sequenced specimen data and with NGS approach per sample (n = 12–17) and for all samples (n = 82).

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    <p>R and P values (Pearson test) of the relationships between the OTU proportions obtained with Sanger-sequenced specimen data and with NGS approach per sample (n = 12–17) and for all samples (n = 82).</p

    Number of OTUs, percentage of tubificids with or without hair setae and IOBS values of each sample obtained with Sanger-sequenced specimen data and NGS approach with uncorrected and corrected sequence abundances.

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    <p>Number of OTUs, percentage of tubificids with or without hair setae and IOBS values of each sample obtained with Sanger-sequenced specimen data and NGS approach with uncorrected and corrected sequence abundances.</p

    Numbers of specimens, of sequenced specimens and of OTUs (Sanger, NGS and total) per sample.

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    <p>Numbers of specimens, of sequenced specimens and of OTUs (Sanger, NGS and total) per sample.</p

    Detection of each OTU in the mixed samples by NGS.

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    <p>A. The heatmap indicates if a taxon present in a mixed sample could be detected in the NGS data (green) or not (blue). The OTUs present in the NGS data of a given sample but not identified by Sanger sequencing are indicated in red. B. The boxplots display the proportions of specimen sequences (blue) and of NGS sequences (red) for each taxon sequenced in at least two mixed samples. OTUs designated by a letter followed by a number are known OTUs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148644#pone.0148644.ref020" target="_blank">20</a>] and OTUs designated by a number in brackets are new; Indet = unidentified.</p

    MOESM1 of DNA barcoding of formalin-fixed aquatic oligochaetes for biomonitoring

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    Additional file 1: Table S1. Performed analyses (formalin fixation for 1 h to 7 days and ethanol fixation) and taxonomic identification per sample. X = analysis performed. Following each taxon name is indicated in brackets how the specimen was identified: 1 = with stereo microscope, 2 = with compound microscope, 3 = with genetic analysis

    Relationships between the proportions of OTUs obtained with Sanger-sequenced specimen data and NGS approach without correction of sequence abundances (left) and after correction (right) per taxon and per sample, for all samples (1–6).

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    <p>Relationships between the proportions of OTUs obtained with Sanger-sequenced specimen data and NGS approach without correction of sequence abundances (left) and after correction (right) per taxon and per sample, for all samples (1–6).</p
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