Search

Clonotype and lineage clustering by ImmunediveRsity . Raw IgG HTS data was subjected to repertoire reconstruction and analysis using ImmunediveRsity. Briefly, ImmunediveRsity assigns IGHV gene to each sequence and clusters them according to IGH clonotypes, which are composed of reads with the same IGHV-IGHJ rearrangement and quasi-identical CDRH3 (≥97 % identity at the nucleotide level). Within a clonotype, one or more lineages can be defined based on SHM pattern (99.5 % identity). IGHV usage frequency can thus be expressed as the proportion of reads using a particular IGHV segment (relative transcription), regardless of clonotype origin, or can be expressed as the proportion of IGH clonotypes or lineages using a particular IGHV segment regarding the total amount of clonotypes or lineages, respectively. (PDF 35 kb)

Higher clonal diversity during acute phase than in the post-convalescent phase, regardless of clinical classification. Rarefaction analysis of acute vs. post-convalescent samples according to clinical status, (A) DWS− (A, green and Pc, dark green) and (B) DWS+ (A, blue and Pc, dark blue); and immune status, (C) primary (A, orange and Pc, brown) and (D) secondary infection (A, light purple and Pc, dark purple) (two-way ANOVA with Bonferroni correction, *p

Somatic hypermutation rates in selected IGHV segments according to clinical and immune status. SHM rates are shown according to clinical status (A, C, and E) and according to immune status (B, D, and F) measured as the proportion of mutations along the VH region [pM-VH (%)]. (A) SHM rates of IGHV1-2 regarding clinical status and (B) immune status. (C) SHM rates of IGHV1-18 regarding clinical status and (D) immune status. (E) SHM rates of IGHV1-69 regarding clinical status and (F) immune status. (DWS− A, green; DWS− Pc, dark green; DWS+ A, blue; DWS+ Pc, dark blue; primary infection acute, orange; primary infection Pc, brown; secondary infection acute, light purple; secondary infection Pc, dark purple) (Kruskal–Wallis test, Dunn’s correction for multiple testing, **p

Absolute number of non-synonymous mutations. Quantitative analysis of non-synonymous somatic hypermutation (DWS− A, green; DWS− Pc, dark green; DWS+ A, blue; DWS+ Pc, dark blue) (Kruskal–Wallis test for non-parametric data with Dunn’s multiple comparison test, *** p

Heat map representation of SHM according to IGHV segment ( rows ) and clinical status ( columns ). Acute DENV infection had significantly lower levels of SHM than post-convalescent and control samples (Mann–Whitney U test for non-parametric data, *** p

Digital CDRH3 “spectratyping” of acute phase DENV infection. CDRH3 “spectratyping” of all segments and according to IGHV1-2, IGHV1-18, and IGHV1-69 segments based on the CDRH3 size of 1280 lineage subsample. Clinical and immune status is shown in upper bars. CDRH3 length (bp) is shown in x axis and lineage relative frequency is shown in y axis (color bars: DWS−, green and DWS+, blue. Upper lines: DWS−, green; DWS+, blue; primary, orange; secondary, purple). (PDF 619 kb)

Days after onset of clinical illness for blood sampling according to clinical status. DWS- A, green; DWS− Pc, dark green; DWS+ A, blue; DWS+ Pc, dark blue. (One-way ANOVA, p > 0.05, not significant). (PDF 16 kb)

Monte Carlo simulation to calculate the probability to sample mB or ASCs. A detailed description of how the simulation model was built, including the sources of information regarding relative amounts of the relevant subpopulations (IgG+ mB cells and ASCs), as well as the relative Ig transcription levels in either subpopulation. (PDF 701 kb)

Preferential IGHV transcription and usage during acute DENV infection. (A). Heat map of hierarchical clustering of the difference between acute minus post-convalescent read relative frequency (ΔA − Pc) per IGHV family. Over-transcription of IGHV family during acute phase is indicated in yellow tones, whereas blue tones indicate under-transcription. Upper rows classify patients according to clinical status (DWS−, green and DWS+, blue) and immune status (primary, orange and secondary, purple) (two-way ANOVA, Bonferroni correction for multiple testing, *p

SHM rates in global and selected IGHV segments of randomly subsampled clonotypes. Each dot represents the percentage of mutations in the largest lineage of 250 randomly sampled clonotypes per individual, classified according to clinical status. (A) Global mutation rates; (B) IGHV1-2; (C) IGHV1-18; and (D) IGHV1-69 (DWS− A, green; DWS− Pc, dark green; DWS+ A, blue; DWS+ Pc, dark blue) (Kruskal–Wallis test, Dunn’s correction for multiple testing, **p

IGHV allelic variation in patients with DENV infection. ImmunediveRsity output data in raw reads were used for allele calling. The matrix depicts each individual and the presence (dark blue) of absence (gray) of IGHV1-2, IGHV1-18, IGHV1-69, and IGHV2-5 alleles. (PDF 22 kb)

SHM rates in global and selected IGHV segments in the ImmunediveRsity -reconstructed repertoire using a CDRH3 identity clustering threshold of 92 %. Each dot represents the percentage of mutations in randomly sampled lineages per individual, classified according to clinical status. (A) Global mutation rates; (B) IGHV1-2; (C) IGHV1-18; and (D) IGHV1-69 (DWS− A, green; DWS− Pc, dark green; DWS+ A, blue; DWS+ Pc, dark blue) (Kruskal–Wallis test, Dunn’s correction for multiple testing, **p

Day 7 post vaccination/day 0 ( D SW d7/ D SW d0) Shannon-Weaver index ratio ( D SW ) for clonotypes (a) and lineages (b) in response to vaccination with TIV and 09 MIV indicates that the 09 MIV vaccine induced an increase in the diversity of the repertoire on day 7 after vaccination, while TIV induced a reduction in the diversity (Mann–Whitney test. **P

Pre- and post-vaccination SHM rates in selected IGHV segments according to vaccination trial. Dots depict mutation rate in VH region (%) per lineage. A total of 670 randomly sampled lineages per individual were selected, filtered according IGHV use, and plotted. (Kruskal-Wallis test. Dunn’s correction for multiple testing. **P

CDRH2 sequence in large IGHV1-69 -expressing V H clonotypes. Sequence logos [55] of CDRH2 (IMGT numbering) of the three largest IGHV1-69 VH clonotypes per individual classified according to (a) homotypic and (b) heterotypic seroconversion, as well as according to (c) no expansion (Δ day 7 – day 0 1 standard deviation (3.4)). (PDF 35 kb)

Serologic analysis of 08/09 TIV and 09 MIV 2009 response. Hemagglutination inhibition assay (HAI) and microneutralization assay (MN) for A(H1N1) 2008, A(H3N2) 2008, and AH1N1pdm 2009 for 08/09 TIV and 09 MIV. Titer is shown in the y axis for pre-immune, 7, 14, and 30 days post vaccination in the x axis. (PDF 357 kb)

Per individual effect of immunization with 09 MIV on the repertoire of peripheral B-lymphocytes. Changes in the relative frequency of use of IGHV segments (Δ day 7 – day 0) for individuals with homotypic seroconversion (blue), and with heterosubtypic seroconversion (red) for 47 IGHV segments. IGHV usage is expressed as the proportion of clonotypes using a particular IGHV segment. (PDF 230 kb)