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1.  De Novo sequencing and transcriptome analysis for Tetramorium bicarinatum: a comprehensive venom gland transcriptome analysis from an ant species 
BMC Genomics  2014;15(1):987.
Arthropod venoms are invaluable sources of bioactive substances with biotechnological application. The limited availability of some venoms, such as those from ants, has restricted the knowledge about the composition and the potential that these biomolecules could represent. In order to provide a global insight on the transcripts expressed in the venom gland of the Brazilian ant species Tetramorium bicarinatum and to unveil the potential of its products, high-throughput approach using Illumina technology has been applied to analyze the genes expressed in active venom glands of this ant species.
A total of 212,371,758 pairs of quality-filtered, 100-base-pair Illumina reads were obtained. The de novo assemblies yielded 36,042 contigs for which 27,873 have at least one predicted ORF among which 59.77% produce significant hits in the available databases. The investigation of the reads mapping toxin class revealed a high diversification with the major part consistent with the classical hymenopteran venom protein signature represented by venom allergen (33.3%), followed by a diverse toxin-expression profile including several distinct isoforms of phospholipase A1 and A2, venom serine protease, hyaluronidase, protease inhibitor and secapin. Moreover, our results revealed for the first time the presence of toxin-like peptides that have been previously identified from unrelated venomous animals such as waprin-like (snakes) and agatoxins (spiders and conus).
The non-toxin transcripts were mainly represented by contigs involved in protein folding and translation, consistent with the protein-secretory function of the venom gland tissue. Finally, about 40% of the generated contigs have no hits in the databases with 25% of the predicted peptides bearing signal peptide emphasizing the potential of the investigation of these sequences as source of new molecules. Among these contigs, six putative novel peptides that show homologies with previously identified antimicrobial peptides were identified.
To the best of our knowledge, this work reports the first large-scale analysis of genes transcribed by the venomous gland of the ant species T. bicarinatum and helps with the identification of Hymenoptera toxin arsenal. In addition, results from this study demonstrate that de novo transcriptome assembly allows useful venom gene expression analysis in a species lacking a genome sequence database.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-987) contains supplementary material, which is available to authorized users.
PMCID: PMC4256838  PMID: 25407482
Tetramorium bicarinatum; Social hymenoptera; Ant; Venom glands; Venom toxins; Hymenopteran allergens; de novo assembly; New generation sequencing; Illumina technology
2.  The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown 
BMC Genomics  2014;15(1):486.
Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology.
The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases.
With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-486) contains supplementary material, which is available to authorized users.
PMCID: PMC4101180  PMID: 24942338
Pycnoporus cinnabarinus; Genome annotation; CAZy; Auxiliary activities; Oxidoreductase; White-rot fungi; Lignocellulose
3.  RNAbrowse: RNA-Seq De Novo Assembly Results Browser 
PLoS ONE  2014;9(5):e96821.
Transcriptome analysis based on a de novo assembly of next generation RNA sequences is now performed routinely in many laboratories. The generated results, including contig sequences, quantification figures, functional annotations and variation discovery outputs are usually bulky and quite diverse. This article presents a user oriented storage and visualisation environment permitting to explore the data in a top-down manner, going from general graphical views to all possible details. The software package is based on biomart, easy to install and populate with local data. The software package is available under the GNU General Public License (GPL) at
PMCID: PMC4019526  PMID: 24823498
4.  Mining for hemicellulases in the fungus-growing termite Pseudacanthotermes militaris using functional metagenomics 
The metagenomic analysis of gut microbiomes has emerged as a powerful strategy for the identification of biomass-degrading enzymes, which will be no doubt useful for the development of advanced biorefining processes. In the present study, we have performed a functional metagenomic analysis on comb and gut microbiomes associated with the fungus-growing termite, Pseudacanthotermes militaris.
Using whole termite abdomens and fungal-comb material respectively, two fosmid-based metagenomic libraries were created and screened for the presence of xylan-degrading enzymes. This revealed 101 positive clones, corresponding to an extremely high global hit rate of 0.49%. Many clones displayed either β-d-xylosidase (EC or α-l-arabinofuranosidase (EC activity, while others displayed the ability to degrade AZCL-xylan or AZCL-β-(1,3)-β-(1,4)-glucan. Using secondary screening it was possible to pinpoint clones of interest that were used to prepare fosmid DNA. Sequencing of fosmid DNA generated 1.46 Mbp of sequence data, and bioinformatics analysis revealed 63 sequences encoding putative carbohydrate-active enzymes, with many of these forming parts of sequence clusters, probably having carbohydrate degradation and metabolic functions. Taxonomic assignment of the different sequences revealed that Firmicutes and Bacteroidetes were predominant phyla in the gut sample, while microbial diversity in the comb sample resembled that of typical soil samples. Cloning and expression in E. coli of six enzyme candidates identified in the libraries provided access to individual enzyme activities, which all proved to be coherent with the primary and secondary functional screens.
This study shows that the gut microbiome of P. militaris possesses the potential to degrade biomass components, such as arabinoxylans and arabinans. Moreover, the data presented suggests that prokaryotic microorganisms present in the comb could also play a part in the degradation of biomass within the termite mound, although further investigation will be needed to clarify the complex synergies that might exist between the different microbiomes that constitute the termitosphere of fungus-growing termites. This study exemplifies the power of functional metagenomics for the discovery of biomass-active enzymes and has provided a collection of potentially interesting biocatalysts for further study.
PMCID: PMC3662619  PMID: 23672637
Functional metagenomics; Fungus-growing termite; Glycoside hydrolases; Hemicellulases; Biomass degradation; Biorefinery
5.  High-density linkage mapping in a pine tree reveals a genomic region associated with inbreeding depression and provides clues to the extent and distribution of meiotic recombination 
BMC Biology  2013;11:50.
The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies.
In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable.
This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.
PMCID: PMC3660193  PMID: 23597128
Unigene; SNP array; Linkage mapping; Segregation distortion; Recombination; Maritime pine; Pinus pinaster
6.  Transcriptional profiling of bud dormancy induction and release in oak by next-generation sequencing 
BMC Genomics  2013;14:236.
In temperate regions, the time lag between vegetative bud burst and bud set determines the duration of the growing season of trees (i.e. the duration of wood biomass production). Dormancy, the period during which the plant is not growing, allows trees to avoid cold injury resulting from exposure to low temperatures. An understanding of the molecular machinery controlling the shift between these two phenological states is of key importance in the context of climatic change. The objective of this study was to identify genes upregulated during endo- and ecodormancy, the two main stages of bud dormancy. Sessile oak is a widely distributed European white oak species. A forcing test on young trees was first carried out to identify the period most likely to correspond to these two stages. Total RNA was then extracted from apical buds displaying endo- and ecodormancy. This RNA was used for the generation of cDNA libraries, and in-depth transcriptome characterization was performed with 454 FLX pyrosequencing technology.
Pyrosequencing produced a total of 495,915 reads. The data were cleaned, duplicated reads removed, and sequences were mapped onto the oak UniGene data. Digital gene expression analysis was performed, with both R statistics and the R-Bioconductor packages (edgeR and DESeq), on 6,471 contigs with read numbers ≥ 5 within any contigs. The number of sequences displaying significant differences in expression level (read abundance) between endo- and ecodormancy conditions ranged from 75 to 161, depending on the algorithm used. 13 genes displaying significant differences between conditions were selected for further analysis, and 11 of these genes, including those for glutathione-S-transferase (GST) and dehydrin xero2 (XERO2) were validated by quantitative PCR.
The identification and functional annotation of differentially expressed genes involved in the “response to abscisic acid”, “response to cold stress” and “response to oxidative stress” categories constitutes a major step towards characterization of the molecular network underlying vegetative bud dormancy, an important life history trait of long-lived organisms.
PMCID: PMC3639946  PMID: 23575249
7.  De Novo Sequencing of Astyanax mexicanus Surface Fish and Pachón Cavefish Transcriptomes Reveals Enrichment of Mutations in Cavefish Putative Eye Genes 
PLoS ONE  2013;8(1):e53553.
Astyanax mexicanus, a teleost species with surface dwelling (surface fish) and cave adapted (cavefish) morphs, is an important model system in evolutionary developmental biology (evodevo). Astyanax cavefish differ from surface fish in numerous traits, including the enhancement of non-visual sensory systems, and the loss of eyes and pigmentation. The genetic bases for these differences are not fully understood as genomic and transcriptomic data are lacking. We here present de novo transcriptome sequencing of embryonic and larval stages of a surface fish population and a cavefish population originating from the Pachón cave using the Sanger method. This effort represents the first large scale sequence and clone resource for the Astyanax research community. The analysis of these sequences show low levels of polymorphism in cavefish compared to surface fish, confirming previous studies on a small number of genes. A high proportion of the genes mutated in cavefish are known to be expressed in the zebrafish visual system. Such a high number of mutations in cavefish putative eye genes may be explained by relaxed selection for vision during the evolution in the absence of light. Based on these sequence differences, we provide a list of 11 genes that are potential candidates for having a role in cavefish visual system degeneration.
PMCID: PMC3541186  PMID: 23326453
8.  NG6: Integrated next generation sequencing storage and processing environment 
BMC Genomics  2012;13:462.
Next generation sequencing platforms are now well implanted in sequencing centres and some laboratories. Upcoming smaller scale machines such as the 454 junior from Roche or the MiSeq from Illumina will increase the number of laboratories hosting a sequencer. In such a context, it is important to provide these teams with an easily manageable environment to store and process the produced reads.
We describe a user-friendly information system able to manage large sets of sequencing data. It includes, on one hand, a workflow environment already containing pipelines adapted to different input formats (sff, fasta, fastq and qseq), different sequencers (Roche 454, Illumina HiSeq) and various analyses (quality control, assembly, alignment, diversity studies,…) and, on the other hand, a secured web site giving access to the results. The connected user will be able to download raw and processed data and browse through the analysis result statistics. The provided workflows can easily be modified or extended and new ones can be added. Ergatis is used as a workflow building, running and monitoring system. The analyses can be run locally or in a cluster environment using Sun Grid Engine.
NG6 is a complete information system designed to answer the needs of a sequencing platform. It provides a user-friendly interface to process, store and download high-throughput sequencing data.
PMCID: PMC3444930  PMID: 22958229
9.  The Medicago Genome Provides Insight into the Evolution of Rhizobial Symbioses 
Young, Nevin D. | Debellé, Frédéric | Oldroyd, Giles E. D. | Geurts, Rene | Cannon, Steven B. | Udvardi, Michael K. | Benedito, Vagner A. | Mayer, Klaus F. X. | Gouzy, Jérôme | Schoof, Heiko | Van de Peer, Yves | Proost, Sebastian | Cook, Douglas R. | Meyers, Blake C. | Spannagl, Manuel | Cheung, Foo | De Mita, Stéphane | Krishnakumar, Vivek | Gundlach, Heidrun | Zhou, Shiguo | Mudge, Joann | Bharti, Arvind K. | Murray, Jeremy D. | Naoumkina, Marina A. | Rosen, Benjamin | Silverstein, Kevin A. T. | Tang, Haibao | Rombauts, Stephane | Zhao, Patrick X. | Zhou, Peng | Barbe, Valérie | Bardou, Philippe | Bechner, Michael | Bellec, Arnaud | Berger, Anne | Bergès, Hélène | Bidwell, Shelby | Bisseling, Ton | Choisne, Nathalie | Couloux, Arnaud | Denny, Roxanne | Deshpande, Shweta | Dai, Xinbin | Doyle, Jeff | Dudez, Anne-Marie | Farmer, Andrew D. | Fouteau, Stéphanie | Franken, Carolien | Gibelin, Chrystel | Gish, John | Goldstein, Steven | González, Alvaro J. | Green, Pamela J. | Hallab, Asis | Hartog, Marijke | Hua, Axin | Humphray, Sean | Jeong, Dong-Hoon | Jing, Yi | Jöcker, Anika | Kenton, Steve M. | Kim, Dong-Jin | Klee, Kathrin | Lai, Hongshing | Lang, Chunting | Lin, Shaoping | Macmil, Simone L | Magdelenat, Ghislaine | Matthews, Lucy | McCorrison, Jamison | Monaghan, Erin L. | Mun, Jeong-Hwan | Najar, Fares Z. | Nicholson, Christine | Noirot, Céline | O’Bleness, Majesta | Paule, Charles R. | Poulain, Julie | Prion, Florent | Qin, Baifang | Qu, Chunmei | Retzel, Ernest F. | Riddle, Claire | Sallet, Erika | Samain, Sylvie | Samson, Nicolas | Sanders, Iryna | Saurat, Olivier | Scarpelli, Claude | Schiex, Thomas | Segurens, Béatrice | Severin, Andrew J. | Sherrier, D. Janine | Shi, Ruihua | Sims, Sarah | Singer, Susan R. | Sinharoy, Senjuti | Sterck, Lieven | Viollet, Agnès | Wang, Bing-Bing | Wang, Keqin | Wang, Mingyi | Wang, Xiaohong | Warfsmann, Jens | Weissenbach, Jean | White, Doug D. | White, Jim D. | Wiley, Graham B. | Wincker, Patrick | Xing, Yanbo | Yang, Limei | Yao, Ziyun | Ying, Fu | Zhai, Jixian | Zhou, Liping | Zuber, Antoine | Dénarié, Jean | Dixon, Richard A. | May, Gregory D. | Schwartz, David C. | Rogers, Jane | Quétier, Francis | Town, Christopher D. | Roe, Bruce A.
Nature  2011;480(7378):520-524.
Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation 1. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Mya). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species 2. Medicago truncatula (Mt) is a long-established model for the study of legume biology. Here we describe the draft sequence of the Mt euchromatin based on a recently completed BAC-assembly supplemented with Illumina-shotgun sequence, together capturing ~94% of all Mt genes. A whole-genome duplication (WGD) approximately 58 Mya played a major role in shaping the Mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the Mt genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max (Gm) and Lotus japonicus (Lj). Mt is a close relative of alfalfa (M. sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the Mt genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox.
PMCID: PMC3272368  PMID: 22089132
10.  RNA-Seq reveals genotype-specific molecular responses to water deficit in eucalyptus 
BMC Genomics  2011;12:538.
In a context of climate change, phenotypic plasticity provides long-lived species, such as trees, with the means to adapt to environmental variations occurring within a single generation. In eucalyptus plantations, water availability is a key factor limiting productivity. However, the molecular mechanisms underlying the adaptation of eucalyptus to water shortage remain unclear. In this study, we compared the molecular responses of two commercial eucalyptus hybrids during the dry season. Both hybrids differ in productivity when grown under water deficit.
Pyrosequencing of RNA extracted from shoot apices provided extensive transcriptome coverage - a catalog of 129,993 unigenes (49,748 contigs and 80,245 singletons) was generated from 398 million base pairs, or 1.14 million reads. The pyrosequencing data enriched considerably existing Eucalyptus EST collections, adding 36,985 unigenes not previously represented. Digital analysis of read abundance in 14,460 contigs identified 1,280 that were differentially expressed between the two genotypes, 155 contigs showing differential expression between treatments (irrigated vs. non irrigated conditions during the dry season), and 274 contigs with significant genotype-by-treatment interaction. The more productive genotype displayed a larger set of genes responding to water stress. Moreover, stress signal transduction seemed to involve different pathways in the two genotypes, suggesting that water shortage induces distinct cellular stress cascades. Similarly, the response of functional proteins also varied widely between genotypes: the most productive genotype decreased expression of genes related to photosystem, transport and secondary metabolism, whereas genes related to primary metabolism and cell organisation were over-expressed.
For the most productive genotype, the ability to express a broader set of genes in response to water availability appears to be a key characteristic in the maintenance of biomass growth during the dry season. Its strategy may involve a decrease of photosynthetic activity during the dry season associated with resources reallocation through major changes in the expression of primary metabolism associated genes. Further efforts will be needed to assess the adaptive nature of the genes highlighted in this study.
PMCID: PMC3248028  PMID: 22047139
12.  Assessment of replicate bias in 454 pyrosequencing and a multi-purpose read-filtering tool 
BMC Research Notes  2011;4:149.
Roche 454 pyrosequencing platform is often considered the most versatile of the Next Generation Sequencing technology platforms, permitting the sequencing of large genomes, the analysis of variations or the study of transcriptomes. A recent reported bias leads to the production of multiple reads for a unique DNA fragment in a random manner within a run. This bias has a direct impact on the quality of the measurement of the representation of the fragments using the reads. Other cleaning steps are usually performed on the reads before assembly or alignment.
PyroCleaner is a software module intended to clean 454 pyrosequencing reads in order to ease the assembly process. This program is a free software and is distributed under the terms of the GNU General Public License as published by the Free Software Foundation. It implements several filters using criteria such as read duplication, length, complexity, base-pair quality and number of undetermined bases. It also permits to clean flowgram files (.sff) of paired-end sequences generating on one hand validated paired-ends file and the other hand single read file.
Read cleaning has always been an important step in sequence analysis. The pyrocleaner python module is a Swiss knife dedicated to 454 reads cleaning. It includes commonly used filters as well as specialised ones such as duplicated read removal and paired-end read verification.
PMCID: PMC3117718  PMID: 21615897
13.  Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak 
BMC Genomics  2010;11:650.
The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity.
We generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser
This genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations.
PMCID: PMC3017864  PMID: 21092232
14.  Non PCR-amplified Transcripts and AFLP fragments as reduced representations of the quail genome for 454 Titanium sequencing 
BMC Research Notes  2010;3:214.
SNP (Single Nucleotide Polymorphism) discovery is now routinely performed using high-throughput sequencing of reduced representation libraries. Our objective was to adapt 454 GS FLX based sequencing methodologies in order to obtain the largest possible dataset from two reduced representations libraries, produced by AFLP (Amplified Fragment Length Polymorphism) for genomic DNA, and EST (Expressed Sequence Tag) for the transcribed fraction of the genome.
The expressed fraction was obtained by preparing cDNA libraries without PCR amplification from quail embryo and brain. To optimize the information content for SNP analyses, libraries were prepared from individuals selected in three quail lines and each individual in the AFLP library was tagged. Sequencing runs produced 399,189 sequence reads from cDNA and 373,484 from genomic fragments, covering close to 250 Mb of sequence in total.
Both methods used to obtain reduced representations for high-throughput sequencing were successful after several improvements.
The protocols may be used for several sequencing applications, such as de novo sequencing, tagged PCR fragments or long fragment sequencing of cDNA.
PMCID: PMC2919564  PMID: 20667075
15.  A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation 
Nucleic Acids Research  2009;37(21):7239-7257.
Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism.
PMCID: PMC2790875  PMID: 19786493
16.  LeARN: a platform for detecting, clustering and annotating non-coding RNAs 
BMC Bioinformatics  2008;9:21.
In the last decade, sequencing projects have led to the development of a number of annotation systems dedicated to the structural and functional annotation of protein-coding genes. These annotation systems manage the annotation of the non-protein coding genes (ncRNAs) in a very crude way, allowing neither the edition of the secondary structures nor the clustering of ncRNA genes into families which are crucial for appropriate annotation of these molecules.
LeARN is a flexible software package which handles the complete process of ncRNA annotation by integrating the layers of automatic detection and human curation.
This software provides the infrastructure to deal properly with ncRNAs in the framework of any annotation project. It fills the gap between existing prediction software, that detect independent ncRNA occurrences, and public ncRNA repositories, that do not offer the flexibility and interactivity required for annotation projects. The software is freely available from the download section of the website
PMCID: PMC2241582  PMID: 18194551

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