Project description:Background: RNA-seq based on short reads generated by next generation sequencing technologies has become the main approach to study differential gene expression. Until now the main applications of this technique have been to study the variation of gene expression in a whole organism, tissue or cell type under different conditions or at different developmental stages. However, RNA-seq also has a great potential to be used in evolutionary studies to investigate gene expression divergence in closely related species. Since the more reliable statistical methods for differential gene expression inference are based on the use of raw read count data, the reference genomes of the species to be compared need to be highly comparable. Results: We show that the published genomes and annotations of the three closely related Drosophila species, D. melanogaster, D. simulans and D. mauritiana, have limitations for inter-specific gene expression studies. This is due to missing gene models in at least one of the genome annotations, unclear orthology assignments and significant length differences in the different species. We propose that published reference genomes should be re-annotated before using them as references for RNA-seq experiments to include as many genes as possible and to account for a potential length bias. For that we present a straight-forward reciprocal re-annotation pipeline that allows to reliably compare the expression for nearly all genes annotated in D. melanogaster. We carried out a RNA-seq experiment in combination with quantitative real-time PCR to confirm that the newly generated gene sets do not result in a high number of false positives as observed with references that still show a clear length difference of gene models between species. Conclusions: We conclude that our reciprocal re-annotation of previously published genomes facilitates the analysis of significantly more genes in an inter-specific differential gene expression study. We propose that the established pipeline can easily be applied to re-annotate other genomes of closely related animals and plants to improve comparative expression analyses.

Project description:In June 2009, the World Health Organization declared the first influenza pandemic of the 21st century, due to the emergence and rapid spread of new swine origin H1N1 influenza A virus. In contrast to seasonal influenza infections, which typically cause morbidity and mortality in the elderly, this virus caused severe infection in young adults and not the elderly. This phenomenon was attributed to the presence of cross-neutralizing antibodies acquired by older individuals from previous exposure to swine origin influenza. However, this hypothesis could not be empirically tested using clinical data. To address this question, we investigated viral replication and the development of the immune response in naï12 years old) and aged (20 to 24 years old) female rhesus macaques infected with A/California/04/2009 (H1N1), one of the circulating pandemic strains in 2009. We compared viral loads as well as the kinetics and magnitude of the adaptive immune response in peripheral blood and bronchoalveolar lavage samples (BAL) collected longitudinally for 99 days post-infection. Although, adult animals exhibited earlier T cell responses in peripheral blood, aged animals generated a robust T cell response with comparable kinetics and magnitude as those observed in young animals in BAL. Moreover, aged animals generated a higher hemagglutination inhibition titer compared to young animals. We also measured the concentration of several cytokines in BAL supernatant. With the exception of IL-8, which was higher in aged animals, we found no differences in IFNa, IFNb, TNFa, IL-1r, IL-6, IL-15, IL-17, or MCP1 levels. Finally, we compared gene expression infection using microarray analysis of BAL samples taken on days 0, 4, 7, 10, and 14 pi. Our analyses revealed that the largest difference in host response between aged and young animals was detected day 4 post-infection, with significant enrichment for genes associated with inflammation, the innate immune response, and T cell activation in aged animals. The ability of aged animals to generate a robust immune response, especially antibody response, following infection with 2009 H1N1 virus could explain the lack of morbidity normally observed with seasonal influenza viruses in this vulnerable population. 16 female rhesus macaques (Macaca Mulatta) 10-12 (Adult) and 20-24 years (Old/Aged) of age were used in these studies. Animals were infected with A/California/04/ 2009 H1N1 using a combinatory of intra-tracheal (4ml), intranasal (0.5 ml/nostril), and conjunctival (0.5 ml/eyelid) routes for a total dose of 7x106 TCID50 dose. Microarray analysis was performed on Bronchoalveolar lavage (BAL) samples collected on days 0, 4, 7, 10 and 14. Note: One of the Day 0 array did not pass QC metrics so for this animal the average of the other Day 0 samples from that group was utilized. At the end of the study animals were released back to the colony.

Project description:Developing B lymphocytes undergo V(D)J recombination to assemble germline V, D, and J gene segments into exons that encode the antigen-binding variable region of immunoglobulin (Ig) heavy (H) and light (L) chains. IgH and IgL chains associate to form the B cell receptor (BCR), which upon antigen binding activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. Ability of V(D)J recombination to generate vast primary B cell repertoires results from combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as HTGTS repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or non-productive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions and also for following antibody affinity maturation processes. We employed high-throughput genome-wide translocation sequencing adapted repertoire sequencing (HTGTS-Rep-seq) to study antibody repertoires. For HTGTS-Rep-seq libraries, we utilize bait coding ends of J segments to identify, in unbiased fashion, mouse IgH DJH repertoires [processed tlx files] along with both productive and non-productive IgH V(D)J repertoires from both pro-B and peripheral B cells [processed xls files of samples 1-18, 21-51]. Similarly, we also identify mouse productive and non-productive Igk repertoires from peripheral B cells [processed xls files of samples 19,20,52-57].

Project description:In June 2009, the World Health Organization declared the first influenza pandemic of the 21st century, due to the emergence and rapid spread of new swine origin H1N1 influenza A virus. In contrast to seasonal influenza infections, which typically cause morbidity and mortality in the elderly, this virus caused severe infection in young adults and not the elderly. This phenomenon was attributed to the presence of cross-neutralizing antibodies acquired by older individuals from previous exposure to swine origin influenza. However, this hypothesis could not be empirically tested using clinical data. To address this question, we investigated viral replication and the development of the immune response in naï12 years old) and aged (20 to 24 years old) female rhesus macaques infected with A/California/04/2009 (H1N1), one of the circulating pandemic strains in 2009. We compared viral loads as well as the kinetics and magnitude of the adaptive immune response in peripheral blood and bronchoalveolar lavage samples (BAL) collected longitudinally for 99 days post-infection. Although, adult animals exhibited earlier T cell responses in peripheral blood, aged animals generated a robust T cell response with comparable kinetics and magnitude as those observed in young animals in BAL. Moreover, aged animals generated a higher hemagglutination inhibition titer compared to young animals. We also measured the concentration of several cytokines in BAL supernatant. With the exception of IL-8, which was higher in aged animals, we found no differences in IFNa, IFNb, TNFa, IL-1r, IL-6, IL-15, IL-17, or MCP1 levels. Finally, we compared gene expression infection using microarray analysis of BAL samples taken on days 0, 4, 7, 10, and 14 pi. Our analyses revealed that the largest difference in host response between aged and young animals was detected day 4 post-infection, with significant enrichment for genes associated with inflammation, the innate immune response, and T cell activation in aged animals. The ability of aged animals to generate a robust immune response, especially antibody response, following infection with 2009 H1N1 virus could explain the lack of morbidity normally observed with seasonal influenza viruses in this vulnerable population.

			Action	DRS
	SRR3045230_1.fastq.gz	Fastqsanger.gz
	SRR3045230_2.fastq.gz	Fastqsanger.gz
	SRR3045231_1.fastq.gz	Fastqsanger.gz
	SRR3045231_2.fastq.gz	Fastqsanger.gz
	SRR3045222.fastq.gz	Fastqsanger.gz

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A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species

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