Disregarding multimappers leads to biases in the functional assessment of NGS data.

BACKGROUND: Standard ChIP-seq and RNA-seq processing pipelines typically disregard sequencing reads whose origin is ambiguous ("multimappers"). This usual practice has potentially important consequences for the functional interpretation of the data: genomic elements belonging to clusters composed of highly similar members are left unexplored. RESULTS: In particular, disregarding multimappers leads to the underrepresentation in epigenetic studies of recently active transposable elements, such as AluYa5, L1HS and SVAs. Furthermore, this common strategy also has implications for transcriptomic analysis: members of repetitive gene families, such the ones including major histocompatibility complex (MHC) class I and II genes, are under-quantified. CONCLUSION: Revealing inherent biases that permeate routine tasks such as functional enrichment analysis, our results underscore the urgency of broadly adopting multimapper-aware bioinformatic pipelines -currently restricted to specific contexts or communities- to ensure the reliability of genomic and transcriptomic studies.

T3E: a tool for characterising the epigenetic profile of transposable elements using ChIP-seq data.

BACKGROUND: Despite the advent of Chromatin Immunoprecipitation Sequencing (ChIP-seq) having revolutionised our understanding of the mammalian genome's regulatory landscape, many challenges remain. In particular, because of their repetitive nature, the sequencing reads derived from transposable elements (TEs) pose a real bioinformatics challenge, to the point that standard analysis pipelines typically ignore reads whose genomic origin cannot be unambiguously ascertained. RESULTS: We show that discarding ambiguously mapping reads may lead to a systematic underestimation of the number of reads associated with young TE families/subfamilies. We also provide evidence suggesting that the strategy of randomly permuting the location of the read mappings (or the TEs) that is often used to compute the background for enrichment calculations at TE families/subfamilies can result in both false positive and negative enrichments. To address these problems, we present the Transposable Element Enrichment Estimator (T3E), a tool that makes use of ChIP-seq data to characterise the epigenetic profile of associated TE families/subfamilies. T3E weights the number of read mappings assigned to the individual TE copies of a family/subfamily by the overall number of genomic loci to which the corresponding reads map, and this is done at the single nucleotide level. In addition, T3E computes ChIP-seq enrichment relative to a background estimated based on the distribution of the read mappings in the input control DNA. We demonstrated the capabilities of T3E on 23 different ChIP-seq libraries. T3E identified enrichments that were consistent with previous studies. Furthermore, T3E detected context-specific enrichments that are likely to pinpoint unexplored TE families/subfamilies with individual TE copies that have been frequently exapted as cis-regulatory elements during the evolution of mammalian regulatory networks. CONCLUSIONS: T3E is a novel open-source computational tool (available for use at: https://github.com/michelleapaz/T3E ) that overcomes some of the pitfalls associated with the analysis of ChIP-seq data arising from the repetitive mammalian genome and provides a framework to shed light on the epigenetics of entire TE families/subfamilies.

KIR2DL4 genetic diversity in a Brazilian population sample: implications for transcription regulation and protein diversity in samples with different ancestry backgrounds.

KIR2DL4 is an important immune modulator expressed in natural killer cells; HLA-G is its main ligand. We have characterized the KIR2DL4 genetic diversity by considering the promoter, all exons, and all introns in a highly admixed Brazilian population sample and by using massively parallel sequencing. We introduce a molecular method to amplify and to sequence the complete KIR2DL4 gene. To avoid the mapping bias and genotype errors commonly observed in gene families, we have developed and validated a bioinformatic pipeline designed to minimize these errors and applied it to survey the variability of 220 individuals from the State of São Paulo, southeastern Brazil. We have also compared the KIR2DL4 genetic diversity in the Brazilian cohort with the diversity previously reported by the 1000Genomes consortium. KIR2DL4 presents high linkage disequilibrium throughout the gene, with coding sequences associated with specific promoters. There are few but divergent promoter haplotypes. We have also detected many new KIR2DL4 sequences, all bearing nucleotide exchanges in introns and encoding previously described proteins. Exons 3 and 4, which encode the external domains, are the most variable. The ancestry background influences the KIR2DL4 allele frequencies and must be considered for association studies regarding KIR2DL4.

Hla-C genetic diversity and evolutionary insights in two samples from Brazil and Benin.

Human leukocyte antigen-C (HLA-C) is a classical HLA class I molecule that binds and presents peptides to cytotoxic T lymphocytes in the cell surface. HLA-C has a dual function because it also interacts with Killer-cell immunoglobulin-like receptors (KIR) receptors expressed in natural killer and T cells, modulating their activity. The structure and diversity of the HLA-C regulatory regions, as well as the relationship among variants along the HLA-C locus, are poorly addressed, and few population-based studies explored the HLA-C variability in the entire gene in different population samples. Here we present a molecular and bioinformatics method to evaluate the entire HLA-C diversity, including regulatory sequences. Then, we applied this method to survey the HLA-C diversity in two population samples with different demographic histories, one highly admixed from Brazil with major European contribution, and one from Benin with major African contribution. The HLA-C promoter and 3'UTR were very polymorphic with the presence of few, but highly divergent haplotypes. These segments also present conserved sequences that are shared among different primate species. Nucleotide diversity was higher in other segments rather than exons 2 and 3, particularly around exon 5 and the second half of the 3'UTR region. We detected evidence of balancing selection on the entire HLA-C locus and positive selection in the HLA-C leader peptide, for both populations. HLA-C motifs previously associated with KIR interaction and expression regulation are similar between both populations. Each allele group is associated with specific regulatory sequences, reflecting the high linkage disequilibrium along the entire HLA-C locus in both populations.

HLA-A promoter, coding, and 3'UTR sequences in a Brazilian cohort, and their evolutionary aspects.

HLA-A is the second most polymorphic locus of the human leucocyte antigen (HLA) complex encoding a key molecule for antigen presentation and NK cell modulation. Many studies have evaluated HLA-A variability in worldwide populations, focusing mainly on exons, but the regulatory segments have been poorly characterized. HLA-A variability is particularly high in the segment encoding the peptide-binding groove (exons 2 and 3), which is related to the antigen presentation function and the balancing selection in these segments. Here we evaluate the genetic diversity of the HLA-A gene considering a continuous segment encompassing the extended promoter (1.5 kb upstream of the first translated ATG), all exons and introns, and the entire 3' untranslated region, by using massively parallel sequencing. To achieve this goal, we used a freely available bioinformatics workflow that optimizes read mapping for HLA genes and defines complete sequences using either the phase among variable sites directly observed in sequencing data and probabilistic models. The HLA-A variability detected in a highly admixed population sample from Brazil shows that the HLA-A regulatory segments present few, but divergent sequences. The regulatory segments are in close association with the coding alleles. Both exons and introns are highly variable. Moreover, patterns of molecular diversity suggest that the promoter, in addition to the coding region, might be under the same selective pressure, but a different scenario arises when it comes to exon 4 and the 3'UTR segment.

Hla-mapper: An application to optimize the mapping of HLA sequences produced by massively parallel sequencing procedures.

A challenging task when more than one HLA gene is evaluated together by second-generation sequencing is to achieve a reliable read mapping. The polymorphic and repetitive nature of HLA genes might bias the read mapping process, usually underestimating variability at very polymorphic segments, or overestimating variability at some segments. To overcome this issue we developed hla-mapper, which takes into account HLA sequences derived from the IPD-IMGT/HLA database and unpublished HLA sequences to apply a scoring system. This comprehends the evaluation of each read pair, addressing them to the most likely HLA gene they were derived from. Hla-mapper provides a reliable map of HLA sequences, allowing accurate downstream analysis such as variant calling, haplotype inference, and allele typing. Moreover, hla-mapper supports whole genome, exome, and targeted sequencing data. To assess the software performance in comparison with traditional mapping algorithms, we used three different simulated datasets to compare the results obtained with hla-mapper, BWA MEM, and Bowtie2. Overall, hla-mapper presented a superior performance, mainly for the classical HLA class I genes, minimizing wrong mapping and cross-mapping that are typically observed when using BWA MEM or Bowtie2 with a single reference genome.

Human leukocyte antigen-G 3' untranslated region polymorphisms are associated with asthma severity.

Asthma is a genetically complex chronic inflammatory airway disorder, and according to disease pathogenesis, clinical manifestations may vary according to asthma severity. A gene region close to the human leukocyte antigen-G (HLA-G) gene was identified as an independent susceptibility marker for asthma. Considering that the HLA-G immune checkpoint molecule may modulate inflammation, we evaluated the diversity of the HLA-G 3' untranslated region (3'UTR) in asthmatic patients stratified according to disease severity. We evaluate the entire HLA-G 3'UTR segment in 115 Brazilian patients stratified into mild (n=29), moderate (n=21) and severe asthmatics (n=65), and in 116 healthy individuals. HLA-G 3'UTR typing was performed using Sanger sequencing. The multiple comparisons among patients stratified according to disease severity revealed several associations; however, after Bonferroni's correction, the following results remained significant: i) the +3010C and +3142G alleles were overrepresented in mild asthma patients when compared to controls; ii) the +3010G and +3142C alleles were overrepresented in severe asthma patients in comparison to patients with mild asthma. In conclusion, the +3010C/G and +3142C/G HLA-G 3'UTR variation sites were differentially associated according to asthma severity.

HLA-G variability and haplotypes detected by massively parallel sequencing procedures in the geographicaly distinct population samples of Brazil and Cyprus.

The HLA-G molecule presents immunomodulatory properties that might inhibit immune responses when interacting with specific Natural Killer and T cell receptors, such as KIR2DL4, ILT2 and ILT4. Thus, HLA-G might influence the outcome of situations in which fine immune system modulation is required, such as autoimmune diseases, transplants, cancer and pregnancy. The majority of the studies regarding the HLA-G gene variability so far was restricted to a specific gene segment (i.e., promoter, coding or 3' untranslated region), and was performed by using Sanger sequencing and probabilistic models to infer haplotypes. Here we propose a massively parallel sequencing (NGS) with a bioinformatics strategy to evaluate the entire HLA-G regulatory and coding segments, with haplotypes inferred relying more on the straightforward haplotyping capabilities of NGS, and less on probabilistic models. Then, HLA-G variability was surveyed in two admixed population samples of distinct geographical regions and demographic backgrounds, Cyprus and Brazil. Most haplotypes (promoters, coding, 3'UTR and extended ones) were detected both in Brazil and Cyprus and were identical to the ones already described by probabilistic models, indicating that these haplotypes are quite old and may be present worldwide.

Induction of protective immunity against Schistosoma mansoni via DNA priming and boosting with the large subunit of calpain (Sm-p80): adjuvant effects of granulocyte-macrophage colony-stimulating factor and interleukin-4.

Considerable morbidity and mortality result from schistosomiasis, an affliction affecting an estimated 200 million people. Although schistosomicidal drugs and other control measures (including public hygiene and snail control) exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. We have targeted a vaccine candidate (large subunit of calpain, Sm-p80) because of its consistent immunogenicity, protective potential, and integral role in surface membrane biogenesis of schistosomes. Since surface membrane renewal appears to be one of the major phenomena employed by schistosomes to evade the host's immune system; an immune response directed against Sm-p80 should render the parasite susceptible to immune clearance from the host by both providing a focus of attack and by potentially impairing the membrane repair process. In the present study, we have employed DNA immunization protocols using Sm-p80 with plasmids encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Sm-p80 by itself provided 39% protection (P = < or =0.0001) against challenge infection in C57BL/6 mice. This protection was increased to 44% (P = < or =0.0001) when the plasmid encoding GM-CSF was coadministered with Sm-p80 DNA. Coinjection of plasmid DNA encoding IL-4 with Sm-p80 DNA yielded a protection level of 42% (P = < or =0.0001). Statistically, the protection conferred by including GM-CSF, but not IL-4, was significantly greater than that when only Sm-p80 was used. Sm-p80 DNA by itself elicited strong responses that include IgG2A and IgG2B antibody isotypes. The introduction of GM-CSF DNA with Sm-p80 DNA led to distinct increases in total IgG and IgG1 titers, whereas the coadministration of IL-4 DNA with Sm-p80 DNA resulted in a slight elevation of IgG1 and IgG3 titers and in some reduction of IgG2A and IgG2B titers. Our data again indicate that Sm-p80 can be an excellent candidate for a schistosomiasis vaccine.

Enhancement of Sm-p80 (large subunit of calpain) induced protective immunity against Schistosoma mansoni through co-delivery of interleukin-2 and interleukin-12 in a DNA vaccine formulation.

Schistosomiasis afflicts an estimated 200 million people in 76 countries and an additional 600 million people are at risk of acquiring this infection. Even though effective anthelmintic treatment and snail eradication control programs exist, the discovery of an effective vaccine still remains the most potentially powerful means of control for this disease. We have concentrated on a vaccine candidate (large subunit of calpain or Sm-p80) because of its potential in conferring protection against challenge infection and its pivotal role in surface membrane biogenesis of schistosomes. Since surface membrane renewal is a major phenomenon employed by hemohelminths to evade host immune system; an immune response directed against Sm-p80 should make the parasite prone to immune clearance from the host by both providing a well-targeted attack and by potentially inhibiting the surface membrane biogenesis process. In the present study, we have utilized DNA immunization protocols using Sm-p80 with plasmids encoding interleukin-2 (IL-2) and interleukin-12 (IL-12). Sm-p80 by itself provided a 39% protection (P