PMC:4502366 / 4787-10093 JSONTXT

{"project":"2_test","denotations":[{"id":"25911226-24695404-43388776","span":{"begin":688,"end":692},"obj":"24695404"},{"id":"25911226-19451168-43388777","span":{"begin":996,"end":1000},"obj":"19451168"},{"id":"25911226-19505943-43388778","span":{"begin":1057,"end":1061},"obj":"19505943"},{"id":"25911226-17906827-43388779","span":{"begin":1158,"end":1162},"obj":"17906827"},{"id":"25911226-2231712-43388780","span":{"begin":1199,"end":1203},"obj":"2231712"},{"id":"25911226-23661685-43388781","span":{"begin":1498,"end":1502},"obj":"23661685"},{"id":"25911226-17906827-43388782","span":{"begin":3126,"end":3130},"obj":"17906827"},{"id":"25911226-22982435-43388783","span":{"begin":3511,"end":3515},"obj":"22982435"},{"id":"25911226-22139921-43388784","span":{"begin":3643,"end":3647},"obj":"22139921"},{"id":"25911226-18033792-43388785","span":{"begin":3826,"end":3830},"obj":"18033792"},{"id":"25911226-22388286-43388786","span":{"begin":4699,"end":4703},"obj":"22388286"}],"text":"Materials and Methods\n\nSequencing and assembly of the T. rathkei mitochondrial genome\nWild-caught specimens of the terrestrial isopod Trachelipus rathkei were obtained from Rice Creek Field Station in Oswego, New York, USA. Genomic DNA was extracted from ventral muscle/nerve, leg, and gonadal tissue from one male and one female, using a DNeasy Blood and Tissue DNA extraction kit (Qiagen). The two samples were barcoded, pooled, and sequenced in a single 2×100 lane on an Illumina HiSeq 2000 at the University at Buffalo, providing a total of ∼140 million sequence read pairs.\nBriefly, adapter sequences were removed and quality filtering was performed using Trimmomatic (Bolger et al. 2014). An initial assembly of all sequence reads was performed using Minia 1.6906 (Chikhi and Rizk 2012; Salikhov and Sacomoto 2013). Putative mitochondrial sequences from this assembly were identified by looking for contigs with relatively high coverage, estimated by mapping reads using bwa (Li and Durbin 2009; Li 2013) and obtaining depth using samtools (Li et al. 2009), and showing high similarity to the Armadillidium vulgare mitochondrial genome (Marcadé et al. 2007) by BLAST searches (Altschul et al. 1990). This search identified a single contig of approximately 13 kb in length with ∼1600× and 680× coverage in the male and female samples, respectively.\nTo obtain a more complete mitochondrial genome assembly, we used an iterative mapping approach, similar to those adopted by others (Hahn et al. 2013), using a custom script. All reads mapping to the putative mitochondrial contig, along with their mates, were re-assembled. Reads mapping to the new assembly were then identified, and this cycle was repeated for 10 iterations. Finally, a separate assembly was constructed for each of the two sequenced individuals, across a range of parameter values. We selected the assembly with the single longest contig as our final reference mitochondrial genome for T. rathkei. For full details of all assembly procedures, please see the Supporting Information, File S1.\n\nSequencing and assembly of the C. convexus mitochondrial genome\nThe C. convexus mitochondrial genome was assembled from data generated as part of the sequencing of the Wolbachia endosymbiont of C. convexus (M. Badawi, B. Moumen, P. Grève, and R. Cordaux, unpublished data). Briefly, gonads of 30 C. convexus female individuals (all sisters), from our laboratory line CCw (derived from individuals caught in Avanton, France, in 1993) were pooled, and total genomic DNA extraction was performed using a standard phenol-chlorophorm protocol. The sample was sequenced by GenoScreen (Lille, France) on 1/4 and 1/8 runs of a 454 GS FLX sequencer using the Titanium chemistry, providing a total of ∼110 Mb of sequence data.\nRead quality was checked with FastQC and reads were assembled de novo (including quality filtering and trimming) using the gsAssemler software implemented in Newbler version 2.6, with default parameters, except a seed step of 1 was used to improve sensitivity. All contigs were mapped against the A. vulgare mitochondrial genome (Marcadé et al. 2007) using MAUVE version 2.3 (Darling et al. 2010) to identify putative mitochondrial contigs. The resulting contigs were then manually resolved into a single contig using the de Bruijn graph of contigs. This contig was approximately 14 kb in length with ∼450× coverage.\n\nAnnotation\nThe T. rathkei mitochondrial genome was initially annotated using the MITOS web server (Bernt et al. 2013), which identifies protein-coding and rRNA genes using homology searches, and a variant of the MITFI algorithm (Juhling et al. 2012) to identify putative tRNA genes. We used the invertebrate mitochondrial genetic code, and the default settings for all other parameters. We also used ARWEN (Laslett and Canback 2008) to provide a complementary set of predicted tRNA genes. Finally, the annotations for the protein-coding and rRNA were manually corrected, aided by BLAST searches against other isopod mitochondrial genes, because in many cases the initial sequences generated by the automated software lacked start or stop codons or were obviously incomplete (e.g., rRNA genes were too short). The C. convexus mitochondrial genome was annotated similarly, using BLAST searches to identify protein-coding and ribosomal RNA genes, and MITFI and ARWEN to identify tRNA genes.\n\nSequence polymorphisms\nFor T. rathkei, the putative mitochondrial reads from each individual were mapped to the reference mitochondrial genome generated above using bwa. For C. convexus, all sequencing reads were mapped to the reference mitochondrial genome generated above using Bowtie2 (Langmead and Salzberg 2012). Full pileup files were generated for each species using the samtools pileup command. All positions with at least 100× coverage in which a nonreference allele was present at a frequency greater than 0.2 were identified as putative polymorphisms.\nSelected polymorphisms were confirmed by Sanger dye-terminator sequencing of the DNA samples used for high-throughput sequencing, as well as two T. rathkei individuals caught in Oswego (United States) and 12 C. convexus individuals originating from various geographic locations (Avanton and Villedaigne in France, and North Carolina in the United States)."}