Socio-demographic indicators, symptoms and co-morbidities of the sampled population.

*<p><b><i>X²</i></b> test.</p

Oligonucleotide sequences used in this study.

*<p>Underlined nucleotides indicate the stop codon for mutagenesis in positions 823–825.</p><p>AdaptAFlx refers to the 454 adaptor A sequence. AdaptAFlx refers to the 454 adaptor B sequence. 454 key indicates 454 library key sequence (TCAG). Nucleotides in bold italics indicate barcodes. Regular font indicates NA gene specific sequence (699–716; 983–1000).</p

Analysis of the relative frequency of Influenza A(H1N1)pdm virus NA mutations identified in this study (A–E) that were reported in the GenBank in 2009 (April to December) in Mexico (n = 181) and the rest of the world (n = 5,032).

<p>Dotted lines represent world-wide isolates, whereas solid lines represent Mexican isolates. Red and blue lines represent alternate alleles. The 912 G/A mutation was not found neither in Morelos nor in Mexican GenBank reports, but was identified elsewhere (<b>F</b>). The absolute (<b>G</b>) and relative (<b>H</b>) number of isolates used for this analysis is shown for Mexico (solid black line) and world-wide isolates (dotted black line). Note that in June, July and August, only 3, 6 and 1 Mexican isolates were reported in the IVR, respectively.</p

Overall metrics and results of the sequencing run.

<p>Na. Not applicable.</p

Sequence coverage plots of the sequenced NA amplicon according to barcode (A) and according to picotiterplate region (B).

<p>Positions of the NA gene are shown in X axis, whereas the number of reads is shown in Y axis. Less variability in read number was obtained according to MID than according to sequencing region, suggesting that the source of variability is during the emPCR amplification.</p

Influenza A (H1N1)pdm NA gene variants identified.

<p>Bold italics: Mutations associated with oseltamivir resistance.</p>&<p>Rounded quotient of the proportional frequency of the haplotype within each barcoded library divided by the actual number (corrected) of amplicon in each barcoded library.</p

Genetic diversity of Influenza A(H1N1)pdm virus in the State of Morelos (2009).

<p>The graph shows the proportion (Y axis) of each of the four genetic groups described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067010#pone-0067010-t003" target="_blank"><b>Table 3</b></a> in each of the 48 libraries, (X axis) ranked according to epidemiological week of the latest individual in the pool. Thus, the earliest cases are seen in the left, whereas the later cases are shown at right. The red dots represent the haplotypes of group IVb, i.e. those haplotypes that contained additional non-synonymous mutations to D248N. Black arrows in the top represent libraries in which individual amplicons were validated by Sanger sequencing. The asterisk (*) indicates those libraries which included an individual from the first wave.</p

Identification by massive sequencing of the IVb.16 (left panel) and IVb.23 (right panel) mutations associated to oseltamivir resistance.

<p><b>A</b>) View of haplotype IVb.16 and haplotype IVb.23 consensus alignment showing the frequency for each individual variant for either haplotype: A/G (black bar) and 823 C/T (red bar) for the H275Y mutation; 740 G/A (green bar) and 742 A/G (black bar) for the S247N mutation. <b>B</b>) Detail of aligned reads. The reference sequence (InDRE 4487) is shown in green. The mutations 823 C/T (left panel) and both 740 G/A and 742 A/G (left panel) are shown in red with a yellow background. <b>C</b>) Representative pyrosequencing flowgrams showing luminous intensity difference at position 823 (left panel), and 740 and 742 (right panel) with respect to the reference (InDRE 4487). <b>D</b>) Sanger sequencing chromatograms corresponding to individuals #791 and #281 belonging to libraries R7B4 (left panel) and R3B2 (right panel) representing the confirmation of genotypic resistance conferred by the H275N and S247N mutations, respectively.</p

Distribution of A(H1N1)pdm influenza cases during May-Nov, 2009 period.

<p><b>A)</b> Overview of the State of Morelos, Mexico. Bars represent the number of cases according to municipality during the first wave (weeks 17–32; blue section of each bar) and during the second wave (weeks 33–47; orange section of each bar). Each individual case is represented in green (haplotypes not associated with oseltamivir resistance) and in red (either the H275Y or the S247N mutations). <b>B)</b> Zoom in of Cuautla city where three clustered cases of the S247N mutation were found.</p

Additional file 3: of Longitudinal analysis of the peripheral B cell repertoire reveals unique effects of immunization with a new influenza virus strain

Sequencing and repertoire mining metrics with ImmunediveRsity [ 30 ] by vaccination trial and by individual. (XLSX 23 kb