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Abstract Background Current high-throughput sequencing platforms provide capacity to sequence multiple samples in parallel. Different samples are labeled by attaching a short sample specific nucleotide sequence, barcode, to each DNA molecule prior pooling them into a mix containing a number of libraries to be sequenced simultaneously. After sequencing, the samples are binned by identifying the barcode sequence within each sequence read. In order to tolerate sequencing errors, barcodes should be sufficiently apart from each other in sequence space. An additional constraint due to both nucleotide usage and basecalling accuracy is that the proportion of different nucleotides should be in balance in each barcode position. The number of samples to be mixed in each sequencing run may vary and this introduces a problem how to select the best subset of available barcodes at sequencing core facility for each sequencing run. There are plenty of tools available for de novo barcode design, but they are not suitable for subset selection. Results We have developed a tool which can be used for three different tasks: 1) selecting an optimal barcode set from a larger set of candidates, 2) checking the compatibility of user-defined set of barcodes, e.g. whether two or more libraries with existing barcodes can be combined in a single sequencing pool, and 3) augmenting an existing set of barcodes. In our approach the selection process is formulated as a minimization problem. We define the cost function and a set of constraints and use integer programming to solve the resulting combinatorial problem. Based on the desired number of barcodes to be selected and the set of candidate sequences given by user, the necessary constraints are automatically generated and the optimal solution can be found. The method is implemented in C programming language and web interface is available at http://ekhidna2.biocenter.helsinki.fi/barcosel. Conclusions Increasing capacity of sequencing platforms raises the challenge of mixing barcodes. Our method allows the user to select a given number of barcodes among the larger existing barcode set so that both sequencing errors are tolerated and the nucleotide balance is optimized. The tool is easy to access via web browser.

Abstract Bacteria of the genus Pectobacterium are economically important plant pathogens that cause soft rot disease on a wide variety of plant species. Here, we report the genome sequence of Pectobacterium carotovorum strain SCC1, a Finnish soft rot model strain isolated from a diseased potato tuber in the early 1980’s. The genome of strain SCC1 consists of one circular chromosome of 4,974,798 bp and one circular plasmid of 5524 bp. In total 4451 genes were predicted, of which 4349 are protein coding and 102 are RNA genes.

Unknown sequences, or gaps, are present in many published genomes across public databases. Gap filling is an important finishing step in de novo genome assembly, especially in large genomes. The gap filling problem is nontrivial and while there are many computational tools partially solving the problem, several have shortcomings as to the reliability and correctness of the output, i.e. the gap filled draft genome. SSPACE-LongRead is a scaffolding tool that utilizes long reads from multiple third-generation sequencing platforms in finding links between contigs and combining them. The long reads potentially contain sequence information to fill the gaps created in the scaffolding, but SSPACE-LongRead currently lacks this functionality. We present an automated pipeline called gapFinisher to process SSPACE-LongRead output to fill gaps after the scaffolding. gapFinisher is based on the controlled use of a previously published gap filling tool FGAP and works on all standard Linux/UNIX command lines. We compare the performance of gapFinisher against two other published gap filling tools PBJelly and GMcloser. We conclude that gapFinisher can fill gaps in draft genomes quickly and reliably. In addition, the serial design of gapFinisher makes it scale well from prokaryote genomes to larger genomes with no increase in the computational footprint.

Soft rot Enterobacteria in the genera Pectobacterium and Dickeya cause rotting of many crop plants. A new Dickeya isolate has been suggested to form a separate species, given the name Dickeya solani. This bacterium is spreading fast and replacing the closely related, but less virulent, potato pathogens. The genome of D. solani isolate D s0432-1 shows highest similarity at the nucleotide level and in synteny to D. dadantii strain 3937, but it also contains three large polyketide/fatty acid/non-ribosomal peptide synthetase clusters that are not present in D. dadantii 3937. These gene clusters may be involved in the production of toxic secondary metabolites, such as oocydin and zeamine. Furthermore, the D. solani genome harbors several specific genes that are not present in other Dickeya and Pectobacterium species and that may confer advantages for adaptation to new environments. In conclusion, the fast spreading of D. solani may be related to the acquisition of new properties that affect its interaction with plants and other microbes in the potato ecosystem.

We characterize allelic and gene expression variation between populations of the Glanville fritillary butterfly (Melitaea cinxia) from two fragmented and two continuous landscapes in northern Europe. The populations exhibit significant differences in their life history traits, e.g. butterflies from fragmented landscapes have higher flight metabolic rate and dispersal rate in the field, and higher larval growth rate, than butterflies from continuous landscapes. In fragmented landscapes, local populations are small and have a high risk of local extinction, and hence the long-term persistence at the landscape level is based on frequent re-colonization of vacant habitat patches, which is predicted to select for increased dispersal rate. Using RNA-seq data and a common garden experiment, we found that a large number of genes (1,841) were differentially expressed between the landscape types. Hexamerin genes, the expression of which has previously been shown to have high heritability and which correlate strongly with larval development time in the Glanville fritillary, had higher expression in fragmented than continuous landscapes. Genes that were more highly expressed in butterflies from newly-established than old local populations within a fragmented landscape were also more highly expressed, at the landscape level, in fragmented than continuous landscapes. This result suggests that recurrent extinctions and re-colonizations in fragmented landscapes select a for specific expression profile. Genes that were significantly up-regulated following an experimental flight treatment had higher basal expression in fragmented landscapes, indicating that these butterflies are genetically primed for frequent flight. Active flight causes oxidative stress, but butterflies from fragmented landscapes were more tolerant of hypoxia. We conclude that differences in gene expression between the landscape types reflect genomic adaptations to landscape fragmentation.

Soft rot disease is economically one of the most devastating bacterial diseases affecting plants worldwide. In this study, we present novel insights into the phylogeny and virulence of the soft rot model Pectobacterium sp. SCC3193, which was isolated from a diseased potato stem in Finland in the early 1980s. Genomic approaches, including proteome and genome comparisons of all sequenced soft rot bacteria, revealed that SCC3193, previously included in the species Pectobacterium carotovorum, can now be more accurately classified as Pectobacterium wasabiae. Together with the recently revised phylogeny of a few P. carotovorum strains and an increasing number of studies on P. wasabiae, our work indicates that P. wasabiae has been unnoticed but present in potato fields worldwide. A combination of genomic approaches and in planta experiments identified features that separate SCC3193 and other P. wasabiae strains from the rest of soft rot bacteria, such as the absence of a type III secretion system that contributes to virulence of other soft rot species. Experimentally established virulence determinants include the putative transcriptional regulator SirB, two partially redundant type VI secretion systems and two horizontally acquired clusters (Vic1 and Vic2), which contain predicted virulence genes. Genome comparison also revealed other interesting traits that may be related to life in planta or other specific environmental conditions. These traits include a predicted benzoic acid/salicylic acid carboxyl methyltransferase of eukaryotic origin. The novelties found in this work indicate that soft rot bacteria have a reservoir of unknown traits that may be utilized in the poorly understood latent stage in planta. The genomic approaches and the comparison of the model strain SCC3193 to other sequenced Pectobacterium strains, including the type strain of P. wasabiae, provides a solid basis for further investigation of the virulence, distribution and phylogeny of soft rot bacteria and, potentially, other bacteria as well.

Molecular tools may greatly improve our understanding of pathogen evolution and epidemiology but technical constraints have hindered the development of genetic resources for parasites compared to free-living organisms. This study aims at developing molecular tools for Podosphaera plantaginis, an obligate fungal pathogen of Plantago lanceolata. This interaction has been intensively studied in the Åland archipelago of Finland with epidemiological data collected from over 4,000 host populations annually since year 2001.A cDNA library of a pooled sample of fungal conidia was sequenced on the 454 GS-FLX platform. Over 549,411 reads were obtained and annotated into 45,245 contigs. Annotation data was acquired for 65.2% of the assembled sequences. The transcriptome assembly was screened for SNP loci, as well as for functionally important genes (mating-type genes and potential effector proteins). A genotyping assay of 27 SNP loci was designed and tested on 380 infected leaf samples from 80 populations within the Åland archipelago. With this panel we identified 85 multilocus genotypes (MLG) with uneven frequencies across the pathogen metapopulation. Approximately half of the sampled populations contain polymorphism. Our genotyping protocol revealed mixed-genotype infection within a single host leaf to be common. Mixed infection has been proposed as one of the main drivers of pathogen evolution, and hence may be an important process in this pathosystem.The developed SNP panel offers exciting research perspectives for future studies in this well-characterized pathosystem. Also, the transcriptome provides an invaluable novel genomic resource for powdery mildews, which cause significant yield losses on commercially important crops annually. Furthermore, the features that render genetic studies in this system a challenge are shared with the majority of obligate parasitic species, and hence our results provide methodological insights from SNP calling to field sampling protocols for a wide range of biological systems.