Heat map showing the order classification rates for optimal choice of primers.

<p>Cells are colored on a gradient from 0% to 100% matches of the primer with the 16S rRNA gene target sequences of the indicated microbial order. The size of the amplicons generated by the various primer pairs are indicated. Furthermore, the percentage of the amplification rate from each primer pair on the various microbial taxa here described is indicated at the top of the heat map.</p

Quantitative data of the 16S rRNA gene sequence datasets used in this study.

*<p>The M dataset stands for mother’s fecal sample dataset; I dataset correspond to the infant’s stool sample dataset.</p

Primers used in this study.

<p>Primers used in this study.</p

Number of sequences per phylotype for each PCR primer pair and sample (stool samples of infants and fecal samples of mothers).

<p>The y axis displays the OTUs at family level detected in this study; each row represents a different OTU. Increasing darkness of the colour scale corresponds to higher estimated relative abundance. At the bottom of the heatmap a representation of the abundance of different phylotypes at the genus level detected by different PCR primer set for each sample has been displayed.</p

Relation between the percentages of 16S rRNA gene sequences obtained using the four different DNA extraction methods and the mixes P1, P2 and P3.

<p>The sequence of <i>B. coccoides</i> and <i>B. producta</i> were indistinguishable and were included in the same group.</p>*<p>Means that only draft genomes are available, thus the 16S copy number is probably underestimated.</p><p>+ Means that no sequenced genomes are available, thus the <i>genera</i> average 16S copy number was used.</p><p>The normalized relative abundance was calculated by division of the relative abundances of every species by their predicted 16S copy number. Results of each sample were normalized so that their sum is 100%.</p

Fecal levels of different microorganisms as analyzed by PCR using different 16S rRNA gene-based primers.

<p>Panel a depicts PCR amplification targeting the 16S rRNA gene of different enteric bifidobacteria as well as key intestinal bacteria using the primers described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068739#pone.0068739-Palmer1" target="_blank">[22] </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068739#pone.0068739-Zhang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068739#pone.0068739-Eckburg1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068739#pone.0068739-Wang1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068739#pone.0068739-Claesson2" target="_blank">[43]</a> and the primer set Probio_Uni/Probio_Rev. Panel b. displays the microbial profile of the amplicons generated by the primer sets described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068739#pone.0068739-Palmer1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068739#pone.0068739-Eckburg1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068739#pone.0068739-Wang1" target="_blank">[33]</a> according to the following taxa: <i>Bifidobacteriaceae</i>, <i>Enterobacteriaceae</i>, <i>Enterococcaceae</i>, <i>Lactobacillales</i>, <i>Bacteroidetes</i> group, and <i>Atopobium</i> group.</p

Rarefaction curves generated for 16S rRNA gene sequences and Principal Coordinate Analysis (PCoA) based on the phylotypes identified from different PCR primer sets as well as from different samples (stool samples of infants and fecal samples of mothers).

<p>Panels a and c display the rarefaction curves and the PCoA from stool samples of infants. Panels b and d show the rarefaction curves and the PCoA from fecal samples of mothers. In panels c and d percentages shown along the axes represent the proportion of dissimilarities captured by the axes. Each symbol represents the 16S rRNA gene sequences from each sample which are displayed in a different colour and shape according to the PCR primer pair. In panels a and b the plots depicted on the left represent the rarefaction curves determined using the PD index whereas the plots show on the right constitute the rarefaction curves obtained using the Chao index. A 95% confidence intervals was added to the rarefaction curves.</p

The genes and encoded products of <i>B. asteroides</i> PRL2011 predicted to be involved in respiration and oxygen damage.

<p>Panel a represents a circular genome atlas of <i>B. asteroides</i> PRL2011 (circle 1) with mapped orthologs (defined as reciprocal best FastA hits with more than 30% identity over at least 80% of both protein lengths) in seven publicly available <i>Bifidobacterium</i> genomes. From the outer circle, circle (2) shows <i>B. breve</i> UCC2003, circle (3) <i>B. animalis</i> subsp. <i>lactis</i> DSM 10140, circle (4) <i>B. longum</i> subsp. <i>infantis</i> ATCC 15697, circle (5) <i>B. dentium</i> Bd1, circle (6) <i>B. longum</i> NCC2705, circle (7) <i>B. bifidum</i> PRL2010, circle (8) <i>B. adolescentis</i> ATCC 15703. Circle(9) illustrates <i>B. asteroides</i> PRL2011 G+C% deviation followed by circle (10) that highlights <i>B. asteroides</i> PRL2011 GC skew (G−C/G+C). Moreover, the outer insets indicate the main genetic loci encoding enzymes involved in respiratory metabolism, which are mapped on the circular genome atlas of <i>B. asteroides</i> PRL2011. Panel b shows a schematic representation of a cell and metabolic pathways for respiration. The different ORFs of <i>B. asteroides</i> PRL2011 encoding the presumed enzymes involved in the respiratory chain are indicated.</p

Genomic diversity in the <i>B. asteroides</i> phylogenetic group with reference to the <i>B. asteroides</i> strain PRL2011 genome.

<p>Panel a shows the comparative genomic hybridization data obtained using different members of the <i>B. asteroides</i> species. Each horizontal row corresponds to a probe on the array, and genes are ordered vertically according to their position on the PRL2011 genome. The columns represent the analysed strains, and strains are indicated by their strain codes. The colour code corresponding to the presence/absence is given at the top right of the figure: the gradient goes from black to yellow to indicate the presence, divergence or absence of a gene sequence. The predicted function of particular genes is shown on the right-hand margin. Black typed descriptions relate to most significant DNA regions that are absent in the investigated strains. Red typed descriptions represent DNA regions that encode enzymes predicted to be involved in respiration in PRL2011. Panel b details the presence (black) or absence (yellow) of key genes predicted to be involved in respiration within the <i>B. asteroides</i> phylogenetic group as well as in other genome sequenced bifidobacteria based on genomic data.</p

Comparative genomic analysis of <i>B. asteroides</i> PRL2011 with other fully sequenced bifidobacterial genomes, as well as <i>Gardnella vaginalis</i>.

<p>Panel a displays a Venn diagram of homologs shared between sequenced bifidobacterial-<i>Gardnella</i> genomes. Panel b shows the percentage of amino acid identity of the top-scoring self-matches for protein-coding genes in the analysed bacteria using the predicted proteome of <i>B. asteroides</i> PRL2011 as a reference. For each bacterium, the deduced protein-coding regions for each gene were compared with those derived from the <i>B. asteroides</i> PRL2011 genome. Panel c depicts a phylogenetic supertree based on the sequences of <i>Bifidobacterium-Gardnella</i> core proteins. Panel d indicates the generated phylogenetic tree based on 16S rRNA gene sequences from the same set of bacteria. Three other members of the <i>Actinobacteria</i> phylum, <i>N. farcinia</i>, <i>T. whipplei</i> and <i>L. xyli</i>, were also included in the analyses depicted in panels c and d, while the trees were rooted using <i>L. salivarius</i> as outgroup.</p