Mutation percentages (≥5%) emerged in HA and NA of X-181 viruses passaged in eggs.

<p>Notes: The percentages obtained from RNA library followed by HiSeq sequencing are presented without parentheses or brackets. The percentages obtained from RNA library followed by MiSeq sequencing are presented in parentheses. The percentages obtained from DNA library followed by HiSeq sequencing are presented in brackets. These mutation percentages were calculated per comparison with the corresponding expanded progenitor. (See accession numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138650#sec002" target="_blank">Materials and Methods</a>.) aa: Amino-acid, nt: Nucleotide.</p

HiSeq sequencing of RNA Library prepared from A/Ca/07/2009 (H1N1) virus.

<p>*: Count of nucleotide location started 20 nucleotides (UTR) before the starting codon AUG.</p><p>Note: These mutation percentages were calculated per comparison of A/California/07/2009 (H1N1) genome with its published sequence. (See accession numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138650#sec002" target="_blank">Materials and Methods</a> paragraph.) aa: Amino acid, nt: Nucleotide.</p

A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H₂O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H₂O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H₂O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.

<p>A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H₂O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H₂O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H₂O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.</p

Percentage of mutations (≥5%) emerged in A/PR/8/34 viruses 1 and 3 genomes that were subjected to RNA library preparation followed by HiSeq sequencing.

<p>Note: These mutation percentages were calculated per comparison of A/PR/8/34 strains 1 and 3 genomes with the corresponding published sequences. (See accession numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138650#sec002" target="_blank">Materials and Methods</a> paragraph.) nt: Nucleotide, aa: Amino-acid.</p

Percentage of mutations (≥5%) emerged in the genome of A/PR/8/34 strain (CBER stock) obtained by sequencing five times its RNA library by HiSeq and MiSeq.

<p>Notes: MiSeq values that differ from HiSeq's values are presented in parentheses. These mutation percentages were calculated per comparison to their published sequences. (See accession numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138650#sec002" target="_blank">Materials and Methods</a> paragraph.) nt: Nucleotide, aa: Amino-acid.</p

Viruses of A/California/07/2009 vaccines, and A/PR/8/34 and wild type A/California/07/2009 viruses used for deep sequencing analysis.

<p>*: A/PR/8/34 virus stock was passaged 13 times in eggs, and the last passaged virus was passaged one time (passage 14) separately in 2 different eggs, the 2 harvested viruses are designated as A/PR/8/34-1 and A/PR/8/34-3.</p><p>Note: CBER: Center for Biologics Evaluation and Research at US Food and Drug Administration.</p

Mutation percentages (≥5%) emerged in HA and NA of X-179A viruses passaged in eggs.

<p>Notes: The percentages obtained from RNA library followed by HiSeq sequencing are presented without parentheses or brackets. The percentages obtained from RNA library followed by MiSeq sequencing are presented in parentheses. The percentages obtained from DNA library followed by HiSeq sequencing are presented in brackets. These mutation percentages were calculated per comparison with the corresponding expanded progenitor. (See accession numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138650#sec002" target="_blank">Materials and Methods</a>.) aa: Amino-acid, nt: Nucleotide.</p

Correlation between fADI and MN methods for Polio I, II and III.

<p>fADI₅₀ were calculated from the Virus Attachment Indexes using a mathematical approximation model (see text for details). fADI showed a significant correlation of virus attachment blocking for polioviruses of serotypes I, II and III with the data of microneutralization assay obtained independently at CBER/FDA using the WHO miconeutralization protocol.</p

Schematic representation of Control ELISA to check for false positive effects in the fADI neutralization assay.

<p>Virus is added to ELISA plates instead of using target cells to check for any false positive readings. Addition of sera (upper arm) or no sera (lower arm) should produce similar reporter signals (fluorescence) indicating the absence or low level of <i>competition</i> of virus-specific labeling antibody with antibodies in test serum. The plates are read on the Synergy HT plate reader (BioTek) after applying the secondary detecting antibody and the fluorescent tag SA-PE. More details are in the text.</p

Schematic representation of fADI neutralization assay.

<p>Incubation of virus with sera (upper arm) or without sera (lower arm) either blocks or does not block virus attachment to target cells, respectively. Virus attached to the cells is detected by virus specific antibody and fluorescent tag SA-PE followed by additional staining cells with diluted Trypan blue and finally reading on Bioplex. The maximum fluorescent signal without serum corresponds to zero blocking, (lower arm), reduced fluorescent signal indicating blocking of the virus with serum antibody (upper arm).</p