Insights into Sex Chromosome Evolution and Aging from the Genome of a Short-Lived Fish.

The killifish Nothobranchius furzeri is the shortest-lived vertebrate that can be bred in the laboratory. Its rapid growth, early sexual maturation, fast aging, and arrested embryonic development (diapause) make it an attractive model organism in biomedical research. Here, we report a draft sequence of its genome that allowed us to uncover an intra-species Y chromosome polymorphism representing-in real time-different stages of sex chromosome formation that display features of early mammalian XY evolution "in action." Our data suggest that gdf6Y, encoding a TGF-ß family growth factor, is the master sex-determining gene in N. furzeri. Moreover, we observed genomic clustering of aging-related genes, identified genes under positive selection, and revealed significant similarities of gene expression profiles between diapause and aging, particularly for genes controlling cell cycle and translation. The annotated genome sequence is provided as an online resource (http://www.nothobranchius.info/NFINgb).

Age-dependent effects of Igf2bp2 on gene regulation, function, and aging of hematopoietic stem cells in mice.

Increasing evidence links metabolism, protein synthesis, and growth signaling to impairments in the function of hematopoietic stem and progenitor cells (HSPCs) during aging. The Lin28b/Hmga2 pathway controls tissue development, and the postnatal downregulation of this pathway limits the self-renewal of adult vs fetal hematopoietic stem cells (HSCs). Igf2bp2 is an RNA binding protein downstream of Lin28b/Hmga2, which regulates messenger RNA stability and translation. The role of Igf2bp2 in HSC aging is unknown. In this study, an analysis of wild-type and Igf2bp2 knockout mice showed that Igf2bp2 regulates oxidative metabolism in HSPCs and the expression of metabolism, protein synthesis, and stemness-related genes in HSCs of young mice. Interestingly, Igf2bp2 expression and function strongly declined in aging HSCs. In young mice, Igf2bp2 deletion mimicked aging-related changes in HSCs, including changes in Igf2bp2 target gene expression and impairment of colony formation and repopulation capacity. In aged mice, Igf2bp2 gene status had no effect on these parameters in HSCs. Unexpectedly, Igf2bp2-deficient mice exhibited an amelioration of the aging-associated increase in HSCs and myeloid-skewed differentiation. The results suggest that Igf2bp2 controls mitochondrial metabolism, protein synthesis, growth, and stemness of young HSCs, which is necessary for full HSC function during young adult age. However, Igf2bp2 gene function is lost during aging, and it appears to contribute to HSC aging in 2 ways: the aging-related loss of Igf2bp2 gene function impairs the growth and repopulation capacity of aging HSCs, and the activity of Igf2bp2 at a young age contributes to aging-associated HSC expansion and myeloid skewing.

Tnfaip2/exoc3-driven lipid metabolism is essential for stem cell differentiation and organ homeostasis.

Lipid metabolism influences stem cell maintenance and differentiation but genetic factors that control these processes remain to be delineated. Here, we identify Tnfaip2 as an inhibitor of reprogramming of mouse fibroblasts into induced pluripotent stem cells. Tnfaip2 knockout impairs differentiation of embryonic stem cells (ESCs), and knockdown of the planarian para-ortholog, Smed-exoc3, abrogates in vivo tissue homeostasis and regeneration-processes that are driven by somatic stem cells. When stimulated to differentiate, Tnfaip2-deficient ESCs fail to induce synthesis of cellular triacylglycerol (TAG) and lipid droplets (LD) coinciding with reduced expression of vimentin (Vim)-a known inducer of LD formation. Smed-exoc3 depletion also causes a strong reduction of TAGs in planarians. The study shows that Tnfaip2 acts epistatically with and upstream of Vim in impairing cellular reprogramming. Supplementing palmitic acid (PA) and palmitoyl-L-carnitine (the mobilized form of PA) restores the differentiation capacity of Tnfaip2-deficient ESCs and organ maintenance in Smed-exoc3-depleted planarians. Together, these results identify a novel role of Tnfaip2 and exoc3 in controlling lipid metabolism, which is essential for ESC differentiation and planarian organ maintenance.

Resting cells rely on the DNA helicase component MCM2 to build cilia.

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues.

Long-lived rodents reveal signatures of positive selection in genes associated with lifespan.

The genetics of lifespan determination is poorly understood. Most research has been done on short-lived animals and it is unclear if these insights can be transferred to long-lived mammals like humans. Some African mole-rats (Bathyergidae) have life expectancies that are multiple times higher than similar sized and phylogenetically closely related rodents. To gain new insights into genetic mechanisms determining mammalian lifespans, we obtained genomic and transcriptomic data from 17 rodent species and scanned eleven evolutionary branches associated with the evolution of enhanced longevity for positively selected genes (PSGs). Indicating relevance for aging, the set of 250 identified PSGs showed in liver of long-lived naked mole-rats and short-lived rats an expression pattern that fits the antagonistic pleiotropy theory of aging. Moreover, we found the PSGs to be enriched for genes known to be related to aging. Among these enrichments were "cellular respiration" and "metal ion homeostasis", as well as functional terms associated with processes regulated by the mTOR pathway: translation, autophagy and inflammation. Remarkably, among PSGs are RHEB, a regulator of mTOR, and IGF1, both central components of aging-relevant pathways, as well as genes yet unknown to be aging-associated but representing convincing functional candidates, e.g. RHEBL1, AMHR2, PSMG1 and AGER. Exemplary protein homology modeling suggests functional consequences for amino acid changes under positive selection. Therefore, we conclude that our results provide a meaningful resource for follow-up studies to mechanistically link identified genes and amino acids under positive selection to aging and lifespan determination.

PosiGene: automated and easy-to-use pipeline for genome-wide detection of positively selected genes.

Many comparative genomics studies aim to find the genetic basis of species-specific phenotypic traits. A prevailing strategy is to search genome-wide for genes that evolved under positive selection based on the non-synonymous to synonymous substitution ratio. However, incongruent results largely due to high false positive rates indicate the need for standardization of quality criteria and software tools. Main challenges are the ortholog and isoform assignment, the high sensitivity of the statistical models to alignment errors and the imperative to parallelize large parts of the software. We developed the software tool PosiGene that (i) detects positively selected genes (PSGs) on genome-scale, (ii) allows analysis of specific evolutionary branches, (iii) can be used in arbitrary species contexts and (iv) offers visualization of the results for further manual validation and biological interpretation. We exemplify PosiGene's performance using simulated and real data. In the simulated data approach, we determined a false positive rate <1%. With real data, we found that 68.4% of the PSGs detected by PosiGene, were shared by at least one previous study that used the same set of species. PosiGene is a user-friendly, reliable tool for reproducible genome-wide identification of PSGs and freely available at https://github.com/gengit/PosiGene.

Naked mole-rat transcriptome signatures of socially suppressed sexual maturation and links of reproduction to aging.

BACKGROUND: Naked mole-rats (NMRs) are eusocially organized in colonies. Although breeders carry the additional metabolic load of reproduction, they are extremely long-lived and remain fertile throughout their lifespan. This phenomenon contrasts the disposable soma theory of aging stating that organisms can invest their resources either in somatic maintenance, enabling a longer lifespan, or in reproduction, at the cost of longevity. Here, we present a comparative transcriptome analysis of breeders vs. non-breeders of the eusocial, long-lived NMR vs. the polygynous and shorter-lived guinea pig (GP). RESULTS: Comparative transcriptome analysis of tissue samples from ten organs showed, in contrast to GPs, low levels of differentiation between sexes in adult NMR non-breeders. After transition into breeders, NMR transcriptomes are markedly sex-specific, show pronounced feedback signaling via gonadal steroids, and have similarities to reproductive phenotypes in African cichlid fish, which also exhibit social status changes between dominant and subordinate phenotypes. Further, NMRs show functional enrichment of status-related expression differences associated with aging. Lipid metabolism and oxidative phosphorylation-molecular networks known to be linked to aging-were identified among most affected gene sets. Remarkably and in contrast to GPs, transcriptome patterns associated with longevity are reinforced in NMR breeders. CONCLUSION: Our results provide comprehensive and unbiased molecular insights into interspecies differences between NMRs and GPs, both in sexual maturation and in the impact of reproduction on longevity. We present molecular evidence that sexual maturation in NMRs is socially suppressed. In agreement with evolutionary theories of aging in eusocial organisms, we have identified transcriptome patterns in NMR breeders that-in contrast to the disposable soma theory of aging-may slow down aging rates and potentially contribute to their exceptional long life- and healthspan.

Species comparison of liver proteomes reveals links to naked mole-rat longevity and human aging.

BACKGROUND: Mammals display a wide range of variation in their lifespan. Investigating the molecular networks that distinguish long- from short-lived species has proven useful to identify determinants of longevity. Here, we compared the livers of young and old long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omics approach. RESULTS: We found that NMR livers display a unique expression pattern of mitochondrial proteins that results in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMRs and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel detoxification pathway linked to longevity and validated it experimentally in the nematode Caenorhabditis elegans. CONCLUSIONS: Our work demonstrates the benefits of integrating proteomic and transcriptomic data to perform cross-species comparisons of longevity-associated networks. Using a multispecies approach, we show at the molecular level that livers of NMRs display progressive age-dependent changes that recapitulate typical signatures of aging despite the negligible senescence and extraordinary longevity of these rodents.

FRAMA: from RNA-seq data to annotated mRNA assemblies.

BACKGROUND: Advances in second-generation sequencing of RNA made a near-complete characterization of transcriptomes affordable. However, the reconstruction of full-length mRNAs via de novo RNA-seq assembly is still difficult due to the complexity of eukaryote transcriptomes with highly similar paralogs and multiple alternative splice variants. Here, we present FRAMA, a genome-independent annotation tool for de novo mRNA assemblies that addresses several post-assembly tasks, such as reduction of contig redundancy, ortholog assignment, correction of misassembled transcripts, scaffolding of fragmented transcripts and coding sequence identification. RESULTS: We applied FRAMA to assemble and annotate the transcriptome of the naked mole-rat and assess the quality of the obtained compilation of transcripts with the aid of publicy available naked mole-rat gene annotations. Based on a de novo transcriptome assembly (Trinity), FRAMA annotated 21,984 naked mole-rat mRNAs (12,100 full-length CDSs), corresponding to 16,887 genes. The scaffolding of 3488 genes increased the median sequence information 1.27-fold. In total, FRAMA detected and corrected 4774 misassembled genes, which were predominantly caused by fusion of genes. A comparison with three different sources of naked mole-rat transcripts reveals that FRAMA's gene models are better supported by RNA-seq data than any other transcript set. Further, our results demonstrate the competitiveness of FRAMA to state of the art genome-based transcript reconstruction approaches. CONCLUSION: FRAMA realizes the de novo construction of a low-redundant transcript catalog for eukaryotes, including the extension and refinement of transcripts. Thereby, results delivered by FRAMA provide the basis for comprehensive downstream analyses like gene expression studies or comparative transcriptomics. FRAMA is available at https://github.com/gengit/FRAMA .

Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons.

Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence.

Genome-wide search for novel human uORFs and N-terminal protein extensions using ribosomal footprinting.

So far, the annotation of translation initiation sites (TISs) has been based mostly upon bioinformatics rather than experimental evidence. We adapted ribosomal footprinting to puromycin-treated cells to generate a transcriptome-wide map of TISs in a human monocytic cell line. A neural network was trained on the ribosomal footprints observed at previously annotated AUG translation initiation codons (TICs), and used for the ab initio prediction of TISs in 5062 transcripts with sufficient sequence coverage. Functional interpretation suggested 2994 novel upstream open reading frames (uORFs) in the 5' UTR, 1406 uORFs overlapping with the coding sequence, and 546 N-terminal protein extensions. The TIS detection method was validated on the basis of previously published alternative TISs and uORFs. Among primates, TICs in newly annotated TISs were significantly more conserved than control codons, both for AUGs and near-cognate codons. The transcriptome-wide map of novel candidate TISs derived as part of the study will shed further light on the way in which human proteome diversity is influenced by alternative translation initiation and regulation.

It's a bit over, is that ok? The subtle surplus from tandem alternative splicing.

Tandem alternative splice sites (TASS) form a defined class of alternative splicing and give rise to mRNA insertion/deletion variants with only small size differences. Previous work has confirmed evolutionary conservation of TASS elements while many cases show only low tissue specificity of isoform ratios. We pinpoint stochasticity and noise as important methodological issues for the dissection of TASS isoform patterns. Resolving such uncertainties, a recent report showed regulation in a cell culture system, with shifts of alternative splicing isoform ratios dependent on cell density. This novel type of regulation affects not only multiple TASS isoforms, but also other alternative splicing classes, in a concerted manner. Here, we discuss how specific regulatory network architectures may be realized through the novel regulation type and highlight the role of differential isoform functions as a key step in order to better understand the functional role of TASS.

Widespread occurrence of alternative splicing at NAGNAG acceptors contributes to proteome plasticity.

Splice acceptors with the genomic NAGNAG motif may cause NAG insertion-deletions in transcripts, occur in 30% of human genes and are functional in at least 5% of human genes. We found five significant biases indicating that their distribution is nonrandom and that they are evolutionarily conserved and tissue-specific. Because of their subtle effects on mRNA and protein structures, these splice acceptors are often overlooked or underestimated, but they may have a great impact on biology and disease.

Statistical inference of allelic imbalance from transcriptome data.

Next-generation sequencing and the availability of high-density genotyping arrays have facilitated an analysis of somatic and meiotic mutations at unprecedented level, but drawing sensible conclusions about the functional relevance of the detected variants still remains a formidable challenge. In this context, the study of allelic imbalance in intermediate RNA phenotypes may prove a useful means to elucidate the likely effects of DNA variants of unknown significance. We developed a statistical framework for the assessment of allelic imbalance in next-generation transcriptome sequencing (RNA-seq) data that requires neither an expression reference nor the underlying nuclear genotype(s), and that allows for allele miscalls. Using extensive simulation as well as publicly available whole-transcriptome data from European-descent individuals in HapMap, we explored the power of our approach in terms of both genotype inference and allelic imbalance assessment under a wide range of practically relevant scenarios. In so doing, we verified a superior performance of our methodology, particularly at low sequencing coverage, compared to the more simplistic approach of completely ignoring allele miscalls. Because the proposed framework can be used to assess somatic mutations and allelic imbalance in one and the same set of RNA-seq data, it will be particularly useful for the analysis of somatic genetic variation in cancer studies.

Comprehensive assessment of sequence variation within the copy number variable defensin cluster on 8p23 by target enriched in-depth 454 sequencing.

BACKGROUND: In highly copy number variable (CNV) regions such as the human defensin gene locus, comprehensive assessment of sequence variations is challenging. PCR approaches are practically restricted to tiny fractions, and next-generation sequencing (NGS) approaches of whole individual genomes e.g. by the 1000 Genomes Project is confined by an affordable sequence depth. Combining target enrichment with NGS may represent a feasible approach. RESULTS: As a proof of principle, we enriched a ~850 kb section comprising the CNV defensin gene cluster DEFB, the invariable DEFA part and 11 control regions from two genomes by sequence capture and sequenced it by 454 technology. 6,651 differences to the human reference genome were found. Comparison to HapMap genotypes revealed sensitivities and specificities in the range of 94% to 99% for the identification of variations.Using error probabilities for rigorous filtering revealed 2,886 unique single nucleotide variations (SNVs) including 358 putative novel ones. DEFB CN determinations by haplotype ratios were in agreement with alternative methods. CONCLUSION: Although currently labor extensive and having high costs, target enriched NGS provides a powerful tool for the comprehensive assessment of SNVs in highly polymorphic CNV regions of individual genomes. Furthermore, it reveals considerable amounts of putative novel variations and simultaneously allows CN estimation.

Accurate prediction of NAGNAG alternative splicing.

Alternative splicing (AS) involving NAGNAG tandem acceptors is an evolutionarily widespread class of AS. Recent predictions of alternative acceptor usage reported better results for acceptors separated by larger distances, than for NAGNAGs. To improve the latter, we aimed at the use of Bayesian networks (BN), and extensive experimental validation of the predictions. Using carefully constructed training and test datasets, a balanced sensitivity and specificity of >or=92% was achieved. A BN trained on the combined dataset was then used to make predictions, and 81% (38/47) of the experimentally tested predictions were verified. Using a BN learned on human data on six other genomes, we show that while the performance for the vertebrate genomes matches that achieved on human data, there is a slight drop for Drosophila and worm. Lastly, using the prediction accuracy according to experimental validation, we estimate the number of yet undiscovered alternative NAGNAGs. State of the art classifiers can produce highly accurate prediction of AS at NAGNAGs, indicating that we have identified the major features of the 'NAGNAG-splicing code' within the splice site and its immediate neighborhood. Our results suggest that the mechanism behind NAGNAG AS is simple, stochastic, and conserved among vertebrates and beyond.

Abundance and size of hyaluronan in naked mole-rat tissues and plasma.

Large amounts of ultra-high molecular weight hyaluronan (HA) have been described as the main cause of cancer resistance in naked mole-rats (Heterocephalus glaber, NMR). Our work examined HA metabolism in these rodents more closely. HA was localized and quantified using HA binding proteins. Its molecular weight was determined using size exclusion chromatography and gel electrophoresis, HA family gene expression using RNAseq analysis, and hyaluronidase activity using zymography. Guinea pigs (Cavia porcellus) and mice (Mus musculus) were used as controls for some experiments. We found that HA localization was similar in NMR, guinea pig, and mouse tissues but NMR had larger amounts and higher molecular weight (maximum, around 2.5 MDa) of HA in serum and almost all tissues tested. We could not find ultra-high molecular weight HA (≥ 4 MDa) in NMR samples, in contrast to previous descriptions. Hyaluronidase-1 had lower expression and activity in NMR than mouse lymph nodes. RNAseq results showed that, among HA family genes, Tnfaip6 and hyaluronidase-3 (Hyal3) were systematically overexpressed in NMR tissues. In conclusion, NMR samples, contrary to expectations, do not harbor ultra-high molecular weight HA, although its amount and average molecular weight are higher in NMR than in guinea pig tissues and serum. Although hyaluronidase expression and activity are lower in NMR than mouse lymph nodes, this not sufficient to explain the presence of high molecular weight HA. A different activity of the NMR HA synthases remains possible. These characteristics, together with extremely high Hyal3 and Tnfaip6 expression, may provide the NMR with a bespoke, and perhaps protective, HA metabolism.

Increased longevity due to sexual activity in mole-rats is associated with transcriptional changes in the HPA stress axis.

Sexual activity and/or reproduction are associated with a doubling of life expectancy in the long-lived rodent genus Fukomys. To investigate the molecular mechanisms underlying this phenomenon, we analyzed 636 RNA-seq samples across 15 tissues. This analysis suggests that changes in the regulation of the hypothalamic-pituitary-adrenal stress axis play a key role regarding the extended life expectancy of reproductive vs. non-reproductive mole-rats. This is substantiated by a corpus of independent evidence. In accordance with previous studies, the up-regulation of the proteasome and so-called 'anti-aging molecules', for example, dehydroepiandrosterone, is linked with enhanced lifespan. On the other hand, several of our results are not consistent with knowledge about aging of short-lived model organisms. For example, we found the up-regulation of the insulin-like growth factor 1/growth hormone axis and several other anabolic processes to be compatible with a considerable lifespan prolongation. These contradictions question the extent to which findings from short-lived species can be transferred to longer-lived ones.

TassDB2 - A comprehensive database of subtle alternative splicing events.

BACKGROUND: Subtle alternative splicing events involving tandem splice sites separated by a short (2-12 nucleotides) distance are frequent and evolutionarily widespread in eukaryotes, and a major contributor to the complexity of transcriptomes and proteomes. However, these events have been either omitted altogether in databases on alternative splicing, or only the cases of experimentally confirmed alternative splicing have been reported. Thus, a database which covers all confirmed cases of subtle alternative splicing as well as the numerous putative tandem splice sites (which might be confirmed once more transcript data becomes available), and allows to search for tandem splice sites with specific features and download the results, is a valuable resource for targeted experimental studies and large-scale bioinformatics analyses of tandem splice sites. Towards this goal we recently set up TassDB (Tandem Splice Site DataBase, version 1), which stores data about alternative splicing events at tandem splice sites separated by 3 nt in eight species. DESCRIPTION: We have substantially revised and extended TassDB. The currently available version 2 contains extensive information about tandem splice sites separated by 2-12 nt for the human and mouse transcriptomes including data on the conservation of the tandem motifs in five vertebrates. TassDB2 offers a user-friendly interface to search for specific genes or for genes containing tandem splice sites with specific features as well as the possibility to download result datasets. For example, users can search for cases of alternative splicing where the proportion of EST/mRNA evidence supporting the minor isoform exceeds a specific threshold, or where the difference in splice site scores is specified by the user. The predicted impact of each event on the protein is also reported, along with information about being a putative target for the nonsense-mediated decay (NMD) pathway. Links are provided to the UCSC genome browser and other external resources. CONCLUSION: TassDB2, available via http://www.tassdb.info, provides comprehensive resources for researchers interested in both targeted experimental studies and large-scale bioinformatics analyses of short distance tandem splice sites.

Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing.

BACKGROUND: The beta-defensin gene cluster (DEFB) at chromosome 8p23.1 is one of the most copy number (CN) variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn's disease), the role of multisite sequence variations (MSV) is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel. RESULTS: Six PCR products spread over approximately 87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From approximately 142,000 reads, approximately 120,000 haplotype calls (HC) were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed. CONCLUSION: Deep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly CN variable loci such as the beta-defensin locus.