A High-density linkage map for Eucalyptus urophylla and E. grandis based on 1,100 F1 progenies genotyped with 6,000 SnP markers : P0474

Next generation sequencing (NGS) technologies and high-throughput genotyping methods offer the possibility to produce dense linkage maps with highly reliable markers which can be anchored on the physical map of the genome. To this end, we developed a 6,000 SNP infinium bead array (Illumina Inc.) for an inter-specific cross between E. urophylla x E. grandis. The SNP discovery was based on whole genome sequencing (100bp paired-end) with theoretical haploid genome coverage of 40X for the E. urophylla parent and 36X for the E. grandis parent. After read alignment on the Eucalyptus grandis BRASUZ1 reference genome sequence, variant calling procedure with GATK software resulted into 6,667,808 SNPs. Applying different selection criteria, three final independent sets of polymorphic SNPs were kept: 2,618 SNPs heterozygous in E. urophylla, 2,524 SNPs heterozygous in E. grandis and 858 double heterozygous SNPs. The array was used to genotype over 1,100 progenies, providing very high accuracy in marker order and genetic distances between loci. We expect an average of 200-250 markers in each of the 11 linkage group of the two parental maps. These high density linkage maps will provide a key tool: to overlay recombination and physical distances of the genome, to map quantitative trait loci (QTL) with high statistical power and to propose positional candidate genes. (Résumé d'auteur

From high throughput 454 GS FLX data analysis process of 16S RNA gene sequences using barcoding to bacterial community exploration

From high throughput 454 GS FLX data analysis process of 16S RNA gene sequences using barcoding to bacterial community exploratio

The ruminal level of trans-10 fatty acids of dairy cows is linked to the composition of bacterial community

The ruminal level of trans-10 fatty acids of dairy cows is linked to the composition of bacterial communit

Identification of laccase genes in Ganoderma boninense draft genome assembly

Ganoderma boninense, a soil born fungus, is the main agent of basal stem rot, one of the most devastating diseases of oil palm (Eleais guinensis). Observation of oil palm infection by G. boninense in natural conditions has shown the fungus capacity to quickly degrade the stem base, leading to important cracks and finally to tree fall. This important degradation of host tissue likely implies lignolitic enzymes, in particular laccase activity. Those enzymes have been well described in several rot disease involving fungus and their role in the pathogenicity of some fungus like the honey mushroom (Armillaria mellea) is well established. In this context, the study of G. boninense wood degradation genes, and in particular of laccase genes, seems to be of key interest to a better understanding of basal stem rot disease. We produced and assembled a draft sequence genome of an Indonesian G. boninense isolate and the draft sequence of a Malaysian isolate transcriptom. The draft genome assembly was annotated ab initio with Augustus software that predicts genes models from genomic sequence. We obtained 22228 gene models, among which 33 showed similarity with laccase. Among these 33 gene models, 25 exhibited the 4 domain laccase signature sequence and seven showed matches with expressed transcripts. Their length, intron number, subcellular addressing and peptide signal are classical of fungal laccases. A phylogenic analysis of G. boninense laccase predicted gene models along with other fungal laccases suggest recent and extensive gene duplication in G. boninense for a laccase clade specific to some polyporales white rot fungi. (Résumé d'auteur

Identification and development of new polymorphic Microsatellite markers for Ganoderma boninense main causal agent of oil palm basal stem rot disease

Ganoderma boninense is a telluric lignicolous basidiomycete and the main causal agent of the basal stem rot, one of the most devastating diseases of oil palm (Elaeis guinensis). While the fight against G. boninense should be a priority in South-East Asia, only scarce information is available about the diversity level of this fungus, and almost nothing is known about its genetic structure and history. In this context, the development of an informative molecular marker set for characterizing G. boninense diversity is a key step to understand the biology of this pathogen. A G. boninense draft genome sequence assembly of 61.5 Mb (from 454 and Illumina sequencing) has been used to identify and develop a set of microsatellite markers (SSR). A total of 652 SSR were identified of which 145 SSR primer sets were developed. These SSR are characterized by motif from 2 to 6 bases long and 5 to 34 repetitions. A total of 97 SSR were successfully amplified on a first small set of G .boninense isolates from Indonesia. Then a population of 48 isolates from several locations in South-East Asia was screened to characterize each locus for allele number, heterozygoty and null allele absence. These results allow us to propose an efficient SSR set to study G boninense in infected oil palm plantations in order to better understand the history of this pathogen. (Résumé d'auteur

Whole-genome, deep pyrosequencing analysis of a duck influenza A virus evolution in swine cells.

We studied the sub-population level evolution of a duck influenza A virus isolate during passage in swine tracheal cells. The complete genomes of the A/mallard/Netherlands/10-Nmkt/1999 strain and its swine cell-passaged descendent were analysed by 454 pyrosequencing with coverage depth ranging from several hundred to several thousand reads at any point. This allowed characterization of defined minority sub-populations of gene segments 2, 3, 4, 5, 7, and 8 present in the original isolate. These minority sub-populations ranged between 9.5% (for segment 2) and 46% (for segment 4) of their respective gene segments in the parental stock. They were likely contributed by one or more viruses circulating within the same area, at the same period and in the same or a sympatric host species. The minority sub-populations of segments 3, 4, and 5 became extinct upon viral passage in swine cells, whereas the minority sub-populations of segments 2, 7 and 8 completely replaced their majority counterparts. The swine cell-passaged virus was therefore a three-segment reassortant and also harboured point mutations in segments 3 and 4. The passaged virus was more homogenous than the parental stock, with only 17 minority single nucleotide polymorphisms present above 5% frequency across the whole genome. Though limited here to one sample, this deep sequencing approach highlights the evolutionary versatility of influenza viruses whereby they exploit their genetic diversity, predilection for mixed infection and reassortment to adapt to a new host environmental niche.This work was supported by a grant from DEFRA and HEFCE under the Veterinary Training and Research Initiative to the Cambridge Infectious Diseases Consortium (VB, LT), BBSRC grants BB/H014306/1 and BB/G00479X/1 (LT), and the French Ministry of Agriculture, INRA and the French Région Midi-Pyrénées (GC, J-LG, VB).This is the accepted version of the original version available at: http://dx.doi.org/10.1016/j.meegid.2013.04.03

Assessment of replicate bias in 454 pyrosequencing and a multi-purpose read-filtering tool

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Findings</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

CX3CR1 Polymorphisms are associated with atopy but not asthma in German children

Chemokines and their receptors are involved in many aspects of immunity. Chemokine CX3CL1, acting via its receptor CX3CR1, regulates monocyte migration and macrophage differentiation as well as T cell-dependent inflammation. Two common, nonsynonymous polymorphisms in CX3CR1 have previously been shown to alter the function of the CX3CL1/CX3CR1 pathway and were suggested to modify the risk for asthma. Using matrix-assisted laser desorption/ionization time-of-flight technology, we genotyped polymorphisms Val249Ile and Thr280Met in a cross-sectional population of German children from Munich (n = 1,159) and Dresden ( n = 1,940). For 249Ile an odds ratio of 0.77 (95% confidence interval 0.63-0.96; p = 0.017) and for 280Met an odds ratio of 0.71 ( 95% confidence interval 0.56-0.89; p = 0.004) were found with atopy in Dresden but not in Munich. Neither polymorphism was associated with asthma. Thus, amino acid changes in CX3CR1 may influence the development of atopy but not asthma in German children. Potentially, other factors such as environmental effects may modify the role of CX3CR1 polymorphisms. Copyright (c) 2007 S. Karger AG, Basel

Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife

Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations.Methodology/Principal Findings : We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance :We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of bacterial prevalence in host populations, and generation of intra-reservoir patterns of bacterial interactions. Lastly, the number of bacterial reads obtained with the 16S-MiSeq could be a good proxy for bacterial prevalence

sigReannot: an oligo-set re-annotation pipeline based on similarities with the Ensembl transcripts and Unigene clusters

<p>Abstract</p> <p>Background</p> <p>Microarray is a powerful technology enabling to monitor tens of thousands of genes in a single experiment. Most microarrays are now using oligo-sets. The design of the oligo-nucleotides is time consuming and error prone. Genome wide microarray oligo-sets are designed using as large a set of transcripts as possible in order to monitor as many genes as possible. Depending on the genome sequencing state and on the assembly state the knowledge of the existing transcripts can be very different. This knowledge evolves with the different genome builds and gene builds. Once the design is done the microarrays are often used for several years. The biologists working in EADGENE expressed the need of up-to-dated annotation files for the oligo-sets they share including information about the orthologous genes of model species, the Gene Ontology, the corresponding pathways and the chromosomal location.</p> <p>Results</p> <p>The results of SigReannot on a chicken micro-array used in the EADGENE project compared to the initial annotations show that 23% of the oligo-nucleotide gene annotations were not confirmed, 2% were modified and 1% were added. The interest of this up-to-date annotation procedure is demonstrated through the analysis of real data previously published.</p> <p>Conclusion</p> <p>SigReannot uses the oligo-nucleotide design procedure criteria to validate the probe-gene link and the Ensembl transcripts as reference for annotation. It therefore produces a high quality annotation based on reference gene sets.</p