Current sequencing methods produce large amounts of data, but genome assemblies based on these data are often woefully incomplete. These incomplete and error-filled assemblies result in many annotation errors, especially in the number of genes present in a genome. In this paper we investigate the magnitude of the problem, both in terms of total gene number and the number of copies of genes in specific families. To do this, we compare multiple draft assemblies against higher-quality versions of the same genomes, using several new assemblies of the chicken genome based on both traditional and next-generation sequencing technologies, as well as published draft assemblies of chimpanzee. We find that upwards of 40% of all gene families are inferred to have the wrong number of genes in draft assemblies, and that these incorrect assemblies both add and subtract genes. Using simulated genome assemblies of Drosophila melanogaster, we find that the major cause of increased gene numbers in draft genomes is the fragmentation of genes onto multiple individual contigs. Finally, we demonstrate the usefulness of RNA-Seq in improving the gene annotation of draft assemblies, largely by connecting genes that have been fragmented in the assembly process.

Pyruvate kinase deficiency (PKD) is the most frequent red blood cell enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Over 250 PKLR-gene mutations have been described, including missense/nonsense, splicing and regulatory mutations, small insertions, small and gross deletions, causing PKD and hemolytic anemia of variable severity. Alu retrotransposons are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. Alu insertions have been associated with a number of human diseases either by disrupting a coding region or a splice signal. Here, we report on two unrelated Middle Eastern patients, both born from consanguineous parents, with transfusion-dependent hemolytic anemia, where sequence analysis revealed a homozygous insertion of AluYb9 within exon 6 of the PKLR gene, causing precipitous decrease of PKLR RNA levels. This Alu element insertion consists a previously unrecognized mechanism underlying pathogenesis of PKD.

Background: All previous genetic testing has failed to identify the genetic cause of syndrome affecting this family for nearly 20 years. The advent of massively parallel next-generation sequencing technologies has provided an opportunity to affordably screen exomes to establish the genetic basis of disease. The utility of whole exome sequencing to identify causative variants of Mendelian disorders has been clearly demonstrated in the research arena. In this report, we describe a family with unique clinical features, including Hirschsprung disease, with a suspected genetic basis. Methods: We employed whole exome sequencing to this case to identify causative mutations. Results: We identified two novel compound heterozygous variants, following an autosomal recessive mode of inheritance, in the candidate gene SALL1, a gene known to cause Townes-Brocks syndrome (TBS) and central nervous system TBS (CNS-TBS). The pathogenicity of the two variants was supported by co-segregation, low frequency, location in mutation hotspot, pathogenicity program predictions, phenotype similarity, and immunohistochemical staining. As previously reported, we observed intrafamilial phenotypic variability among the affected individuals and they may represent an expansion of TBS, CNS-TBS or a new CNS-TBS-like syndrome by observing atypical features in affected individuals including mental retardation, developmental delay, tracheal anomalies, Hirschsprung disease and cleft lip and palate. Conclusion: Solving this case brought a new view of the genetics of TBS and its relationship with SALL1. Another lesson learned is that advanced technologies have a profound impact on old, unsolved cases such as the one presented here. Thus, we further demonstrated the utility of exome sequencing in the research arena.

No abstract