2011 German Escherichia coli outbreak: Alignment-free whole-genome phylogeny by feature frequency profiles

Accuracy of SNP-based whole-genome phylogeny reconstruction relies heavily on quality of sequence alignment which is particularly hindered by poorly assembled genomes. Alignment-free methods might provide additional insights. Here, we constructed a whole-genome phylogeny of 9 E.coli isolates from the 2011 German outbreak against existing E. coli genomes using the alignment-free feature frequency profile method. In addition, we looked for gene elements that distinguish the outbreak group from the other E. coli strains and possibly accounted for the emergence of the outbreak isolates using the distinguishing feature analysis

2011 German Escherichia coli O104:H4 outbreak: whole-genome phylogeny without alignment

<p>Abstract</p> <p>Background</p> <p>A large-scale <it>Escherichia coli </it>O104:H4 outbreak occurred in Germany from May to July 2011, causing numerous cases of hemolytic-uremic syndrome (HUS) and deaths. Genomes of ten outbreak isolates and a historical O104:H4 strain isolated in 2001 were sequenced using different new generation sequencing platforms. Phylogenetic analyses were performed using various approaches which either are not genome-wide or may be subject to errors due to poor sequence alignment. Also, detailed pathogenicity analyses on the 2001 strain were not available.</p> <p>Findings</p> <p>We reconstructed the phylogeny of <it>E. coli </it>using the genome-wide and alignment-free feature frequency profile method and revealed the 2001 strain to be the closest relative to the 2011 outbreak strain among all available <it>E. coli </it>strains at present and confirmed findings from previous alignment-based phylogenetic studies that the HUS-causing O104:H4 strains are more closely related to typical enteroaggregative <it>E. coli </it>(EAEC) than to enterohemorrhagic <it>E. coli</it>. Detailed re-examination of pathogenicity-related virulence factors and secreted proteins showed that the 2001 strain possesses virulence factors shared between typical EAEC and the 2011 outbreak strain.</p> <p>Conclusions</p> <p>Our study represents the first attempt to elucidate the whole-genome phylogeny of the 2011 German outbreak using an alignment-free method, and suggested a direct line of ancestry leading from a putative EAEC-like ancestor through the 2001 strain to the 2011 outbreak strain.</p

2011 German Escherichia coli outbreak: Prophage analysis of close-assembled TY2482 against 55989 using PHAST

Using the Hiseq data of the German E. coli outbreak isolate TY2482, preliminary prophage analyses have been performed by some researchers previously. With the closed assembly of the same isolate being available, another round of analysis might help in resolving questions that remain unclear due to the incompleteness of the dataset

DNA methods for identification of Chinese medicinal materials

As adulterated and substituted Chinese medicinal materials are common in the market, therapeutic effectiveness of such materials cannot be guaranteed. Identification at species-, strain- and locality-levels, therefore, is required for quality assurance/control of Chinese medicine. This review provides an informative introduction to DNA methods for authentication of Chinese medicinal materials. Technical features and examples of the methods based on sequencing, hybridization and polymerase chain reaction (PCR) are described and their suitability for different identification objectives is discussed

Genome sequence and genetic linkage analysis of Shiitake mushroom _Lentinula edodes_

_Lentinula edodes_ (Shiitake/Xianggu) is an important cultivated mushroom. Understanding the genomics and functional genomics of _L. edodes_ allows us to improve its cultivation and quality. Genome sequence is a key to develop molecular genetic markers for breeding and genetic manipulation. We sequenced the genome of _L. edodes_ monokaryon L54A using Roche 454 and ABI SOLiD genome sequencing. Sequencing reads of about 1400Mb were de novo assembled into a 40.2 Mb genome sequence. We compiled the genome sequence into a searchable database with which we have been annotating the genes and analyzing the metabolic pathways. In addition, we have been using many molecular techniques to analyze genes differentially expressed during development. Gene ortholog groups of _L. edodes_ genome sequence compared across genomes of several fungi including mushrooms identified gene families unique to mushroom-forming fungi. We used a mapping population of haploid basidiospores of dikaryon L54 for genetic linkage analysis. High-quality variations such as single nucleotide polymorphisms, insertions, and deletions of the mapping population formed a high-density genetic linkage map. We compared the linkage map to the _L. edodes_ L54A genome sequence and located selected quantitative trait loci. The Shiitake community will benefit from these resources for genetic studies and breeding.&#xd;&#xa

A cost-effective and universal strategy for complete prokaryotic genomic sequencing proposed by computer simulation

Background: Pyrosequencing techniques allow scientists to perform prokaryotic genome sequencing to achieve the draft genomic sequences within a few days. However, the assemblies with shotgun sequencing are usually composed of hundreds of contigs. A further multiplex PCR procedure is needed to fill all the gaps and link contigs into complete chromosomal sequence, which is the basis for prokaryotic comparative genomic studies. In this article, we study various pyrosequencing strategies by simulated assembling from 100 prokaryotic genomes. Findings. Simulation study shows that a single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) can produce: 1) ∼90% of 100 assemblies with 99.99%; 4) average false gene duplication rate is < 0.7%; 5) average false gene loss rate is < 0.4%. Conclusions: A single end 454 Jr. run combined with a paired end 454 Jr. run (8 kb library) is a cost-effective way for prokaryotic whole genome sequencing. This strategy provides solution to produce high quality draft assemblies for most of prokaryotic organisms within days. Due to the small number of assembled scaffolds, the following multiplex PCR procedure (for gap filling) would be easy. As a result, large scale prokaryotic whole genome sequencing projects may be finished within weeks. © 2012 Jiang et al; BioMed Central Ltd.published_or_final_versio

5'-Serial Analysis of Gene Expression studies reveal a transcriptomic switch during fruiting body development in Coprinopsis cinerea

Abstract: Background: The transition from the vegetative mycelium to the primordium during fruiting body development is the most complex and critical developmental event in the life cycle of many basidiomycete fungi. Understanding the molecular mechanisms underlying this process has long been a goal of research on basidiomycetes. Large scale assessment of the expressed transcriptomes of these developmental stages will facilitate the generation of a more comprehensive picture of the mushroom fruiting process. In this study, we coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development. Results: We evaluated the expression of >3,000 genes in the two respective growth stages and discovered that almost one-third of these genes were preferentially expressed in either stage. This identified a significant turnover of the transcriptome during the course of fruiting body development. Additionally, we annotated more than 79,000 transcription start sites (TSSs) based on the transcriptomes of the mycelium and stage 1 primoridum stages. Patterns of enrichment based on gene annotations from the GO and KEGG databases indicated that various structural and functional protein families were uniquely employed in either stage and that during primordial growth, cellular metabolism is highly up-regulated. Various signaling pathways such as the cAMP-PKA, MAPK and TOR pathways were also identified as up-regulated, consistent with the model that sensing of nutrient levels and the environment are important in this developmental transition. More than 100 up-regulated genes were also found to be unique to mushroom forming basidiomycetes, highlighting the novelty of fruiting body development in the fungal kingdom. Conclusions: We implicated a wealth of new candidate genes important to early stages of mushroom fruiting development, though their precise molecular functions and biological roles are not yet fully known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea

Constructing a new integrated genetic linkage map and mapping quantitative trait loci for vegetative mycelium growth rate in Lentinula edodes

The most saturated linkage map for Lentinula edodes to date was constructed based on a mono-. karyotic population of 146 single spore isolates (SSIs) using sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), insertion deletion (InDel) markers, and the mating-type loci. Five hundred and twenty-four markers were located on 13 linkage groups (LGs). The map spanned a total length of 1006.1 cM, with an average marker spacing of 2.0 cM. Quantitative trait loci (QTLs) mapping was utilized to uncover the loci regulating and controlling the vegetative mycelium growth rate on various synthetic media, and complex medium for commercial cultivation of L. edodes. Two and 13 putative QTLs, identified respectively in the monokaryotic population and two testcross dikaryotic populations, were mapped on seven different LGs. Several vegetative mycelium growth rate-related QTLs uncovered here were clustered on LG4 (Qmgr1, Qdgr1, Qdgr2 and Qdgr9) and LG6 (Qdgr3, Qdgr4 and Qdgr5), implying the presence of main genomic areas responsible for growth rate regulation and control. The QTL hotspot region on LG4 was found to be in close proximity to:the region containing the mating-type A (MAT-A) locus. Moreover, Qdgr2 on LG4 was detected on different media, contributing 8.07%-23.71% of the phenotypic variation. The present study provides essential information for QTL mapping and marker-assisted selection (MAS) in L. edodes. (C) 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.The most saturated linkage map for Lentinula edodes to date was constructed based on a mono-. karyotic population of 146 single spore isolates (SSIs) using sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), insertion deletion (InDel) markers, and the mating-type loci. Five hundred and twenty-four markers were located on 13 linkage groups (LGs). The map spanned a total length of 1006.1 cM, with an average marker spacing of 2.0 cM. Quantitative trait loci (QTLs) mapping was utilized to uncover the loci regulating and controlling the vegetative mycelium growth rate on various synthetic media, and complex medium for commercial cultivation of L. edodes. Two and 13 putative QTLs, identified respectively in the monokaryotic population and two testcross dikaryotic populations, were mapped on seven different LGs. Several vegetative mycelium growth rate-related QTLs uncovered here were clustered on LG4 (Qmgr1, Qdgr1, Qdgr2 and Qdgr9) and LG6 (Qdgr3, Qdgr4 and Qdgr5), implying the presence of main genomic areas responsible for growth rate regulation and control. The QTL hotspot region on LG4 was found to be in close proximity to:the region containing the mating-type A (MAT-A) locus. Moreover, Qdgr2 on LG4 was detected on different media, contributing 8.07%-23.71% of the phenotypic variation. The present study provides essential information for QTL mapping and marker-assisted selection (MAS) in L. edodes. (C) 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved

High-Density Transcriptional Initiation Signals Underline Genomic Islands in Bacteria

Genomic islands (GIs), frequently associated with the pathogenicity of bacteria and having a substantial influence on bacterial evolution, are groups of “alien” elements which probably undergo special temporal–spatial regulation in the host genome. Are there particular hallmark transcriptional signals for these “exotic” regions? We here explore the potential transcriptional signals that underline the GIs beyond the conventional views on basic sequence composition, such as codon usage and GC property bias. It showed that there is a significant enrichment of the transcription start positions (TSPs) in the GI regions compared to the whole genome of Salmonella enterica and Escherichia coli. There was up to a four-fold increase for the 70% GIs, implying high-density TSPs profile can potentially differentiate the GI regions. Based on this feature, we developed a new sliding window method GIST, Genomic-island Identification by Signals of Transcription, to identify these regions. Subsequently, we compared the known GI-associated features of the GIs detected by GIST and by the existing method Islandviewer to those of the whole genome. Our method demonstrates high sensitivity in detecting GIs harboring genes with biased GI-like function, preferred subcellular localization, skewed GC property, shorter gene length and biased “non-optimal” codon usage. The special transcriptional signals discovered here may contribute to the coordinate expression regulation of foreign genes. Finally, by using GIST, we detected many interesting GIs in the 2011 German E. coli O104:H4 outbreak strain TY-2482, including the microcin H47 system and gene cluster ycgXEFZ-ymgABC that activates the production of biofilm matrix. The aforesaid findings highlight the power of GIST to predict GIs with distinct intrinsic features to the genome. The heterogeneity of cumulative TSPs profiles may not only be a better identity for “alien” regions, but also provide hints to the special evolutionary course and transcriptional regulation of GI regions