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      Marteilia refringens and Marteilia pararefringens sp. nov. are distinct parasites of bivalves and have different European distributions

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          Abstract

          Marteilia refringens causes marteiliosis in oysters, mussels and other bivalve molluscs. This parasite previously comprised two species, M. refringens and Marteilia maurini, which were synonymized in 2007 and subsequently referred to as M. refringens ‘O-type’ and ‘M-type’. O-type has caused mass mortalities of the flat oyster Ostrea edulis. We used high throughput sequencing and histology to intensively screen flat oysters and mussels ( Mytilus edulis) from the UK, Sweden and Norway for infection by both types and to generate multi-gene datasets to clarify their genetic distinctiveness. Mussels from the UK, Norway and Sweden were more frequently polymerase chain reaction (PCR)-positive for M-type (75/849) than oysters (11/542). We did not detect O-type in any northern European samples, and no histology-confirmed Marteilia-infected oysters were found in the UK, Norway and Sweden, even where co-habiting mussels were infected by the M-type. The two genetic lineages within ‘ M. refringens’ are robustly distinguishable at species level. We therefore formally define them as separate species: M. refringens (previously O-type) and Marteilia pararefringens sp. nov. (M-type). We designed and tested new Marteilia-specific PCR primers amplifying from the 3’ end of the 18S rRNA gene through to the 5.8S gene, which specifically amplified the target region from both tissue and environmental samples.

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          FLASH: fast length adjustment of short reads to improve genome assemblies.

          Next-generation sequencing technologies generate very large numbers of short reads. Even with very deep genome coverage, short read lengths cause problems in de novo assemblies. The use of paired-end libraries with a fragment size shorter than twice the read length provides an opportunity to generate much longer reads by overlapping and merging read pairs before assembling a genome. We present FLASH, a fast computational tool to extend the length of short reads by overlapping paired-end reads from fragment libraries that are sufficiently short. We tested the correctness of the tool on one million simulated read pairs, and we then applied it as a pre-processor for genome assemblies of Illumina reads from the bacterium Staphylococcus aureus and human chromosome 14. FLASH correctly extended and merged reads >99% of the time on simulated reads with an error rate of <1%. With adequately set parameters, FLASH correctly merged reads over 90% of the time even when the reads contained up to 5% errors. When FLASH was used to extend reads prior to assembly, the resulting assemblies had substantially greater N50 lengths for both contigs and scaffolds. The FLASH system is implemented in C and is freely available as open-source code at http://www.cbcb.umd.edu/software/flash. t.magoc@gmail.com.
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            Reconstructing mitochondrial genomes directly from genomic next-generation sequencing reads—a baiting and iterative mapping approach

            We present an in silico approach for the reconstruction of complete mitochondrial genomes of non-model organisms directly from next-generation sequencing (NGS) data—mitochondrial baiting and iterative mapping (MITObim). The method is straightforward even if only (i) distantly related mitochondrial genomes or (ii) mitochondrial barcode sequences are available as starting-reference sequences or seeds, respectively. We demonstrate the efficiency of the approach in case studies using real NGS data sets of the two monogenean ectoparasites species Gyrodactylus thymalli and Gyrodactylus derjavinoides including their respective teleost hosts European grayling (Thymallus thymallus) and Rainbow trout (Oncorhynchus mykiss). MITObim appeared superior to existing tools in terms of accuracy, runtime and memory requirements and fully automatically recovered mitochondrial genomes exceeding 99.5% accuracy from total genomic DNA derived NGS data sets in <24 h using a standard desktop computer. The approach overcomes the limitations of traditional strategies for obtaining mitochondrial genomes for species with little or no mitochondrial sequence information at hand and represents a fast and highly efficient in silico alternative to laborious conventional strategies relying on initial long-range PCR. We furthermore demonstrate the applicability of MITObim for metagenomic/pooled data sets using simulated data. MITObim is an easy to use tool even for biologists with modest bioinformatics experience. The software is made available as open source pipeline under the MIT license at https://github.com/chrishah/MITObim.
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                Author and article information

                Journal
                Parasitology
                Parasitology
                PAR
                Parasitology
                Cambridge University Press (Cambridge, UK )
                0031-1820
                1469-8161
                September 2018
                11 June 2018
                : 145
                : 11
                : 1483-1492
                Affiliations
                [1 ]Pathology and Microbial Systematics Theme, Centre for Environment, Fisheries and Aquaculture Science (Cefas), Weymouth Laboratory , Weymouth, Dorset DT4 8UB, UK
                [2 ]Biosciences, College of Life and Environmental Sciences, Stocker Road, University of Exeter , Exeter EX4 4QD, UK
                [3 ]Department of Life Sciences, The Natural History Museum , Cromwell Road, SW7 5BD, London, UK
                [4 ]Division of fish, Department of animal health and antimicrobial strategies, National Veterinary Institute (SVA) , Sweden
                [5 ]Institute of Marine Research , PO. Box 1870, Nordnes, 5817 Bergen, Norway
                [6 ]Centro de Investigacións Mariñas, Consellería do Mar da Xunta de Galicia , 36620 Vilanova de Arousa, Spain
                [7 ]Department of Life Sciences, University of Alcalá , 28871 Alcalá de Henares, Spain
                [8 ]Institut Français de Recherche pour l'Exploitation de la Mer (Ifremer) , Laboratoire de Génétique et Pathologie des Mollusques Marins, Avenue de Mus de Loup, 17390 La Tremblade, France
                Author notes
                Author for correspondence: Grant D. Stentiford, E-mail: grant.stentiford@ 123456cefas.co.uk
                Article
                S003118201800063X 00063
                10.1017/S003118201800063X
                6137380
                29886855
                65a49548-89d4-4f11-9028-0775d1f25e22
                © Crown Copyright and Cambridge University Press 2018

                This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 19 July 2017
                : 16 January 2018
                : 08 February 2018
                Page count
                Figures: 3, Tables: 3, References: 67, Pages: 10
                Categories
                Research Article

                Parasitology
                marteilia refringens,marteilia pararefringens,its1 rdna,igs rdna,paramyxida,ascetosporea,mytilus edulis,ostrea edulis

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