De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology

Quality control and data-handling in multicentre studies: the case of the Multicentre Project for Tuberculosis Research

<p>Abstract</p> <p>Background</p> <p>The Multicentre Project for Tuberculosis Research (MPTR) was a clinical-epidemiological study on tuberculosis carried out in Spain from 1996 to 1998. In total, 96 centres scattered all over the country participated in the project, 19935 "possible cases" of tuberculosis were examined and 10053 finally included. Data-handling and quality control procedures implemented in the MPTR are described.</p> <p>Methods</p> <p>The study was divided in three phases: 1) preliminary phase, 2) field work 3) final phase. Quality control procedures during the three phases are described. Results: Preliminary phase: a) organisation of the research team; b) design of epidemiological tools; training of researchers. Field work: a) data collection; b) data computerisation; c) data transmission; d) data cleaning; e) quality control audits; f) confidentiality. Final phase: a) final data cleaning; b) final analysis.</p> <p>Conclusion</p> <p>The undertaking of a multicentre project implies the need to work with a heterogeneous research team and yet at the same time attain a common goal by following a homogeneous methodology. This demands an additional effort on quality control.</p

Selective depletion of CD14+ CD16+ monocytes by glucocorticoid therapy

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on many cells of the body, including monocytes. Here we show that a 5-day course of high dose GC therapy differentially affected the CD14++ and the CD14+ CD16+ monocyte subpopulations in 10 patients treated for multiple sclerosis. While the classical (CD14++) monocytes exhibited a substantial increase from 495 ± 132 to 755 ± 337 cells/μl, the CD14+ CD16+ monocytes responded with a pronounced decrease from 36 ± 15 to 2 ± 3 cells/μl (P < 0.001). In 4/10 patients the CD14+ CD16+ monocytes fell below detection limits (< 0.2 cells/μl). This observation was confirmed when the CD14+ CD16+ monocytes were identified by virtue of their low CD33 expression as these cells decreased as well. After discontinuation of GC therapy the CD14+ CD16+ monocytes reappeared and reached normal levels after 1 week. The profound depletion of CD14+ CD16+ monocytes by GC as described here is a novel effect of GC action in vivo and may contribute to GC-mediated immunosuppression. Determination of the number of this monocyte subset may also serve to monitor the effectiveness of GC therapy in patients requiring immunosuppressive treatment