Clc genomics workbench version 7

DNA was extracted from a pure bacterial culture from all viable strains with a commercial kit according to the manufacturer’s instructions (MasterPure Complete DNA and RNA Purification Kit, Epicentre, distributed by Biozym Scientific, Hessisch Oldendorf, Germany) and DNA from all strains was subjected to genome sequencing. De novo assembly was performed with CLC Genomics Workbench, Version 7.5 (CLC Bio, Aarhus, Denmark). For automatic annotation we used the RAST Server: Rapid Annotations using Subsystems Technology [32 (link)]. Sequence analysis from non-published genomes and calculation of G/C contents were carried out with Geneious (v. 8.1.3; Biomatters, Auckland, NZ) [33 (link)]. Nucleotide sequences of partial 16S rRNA genes and of groEL, recA and gyrB genes were aligned by using MAFFT [34 (link)] (MAFFT v7.017, implemented in Geneious). Maximum likelihood phylogenies and trees were estimated (100 bootstrap replicates) and visualized with PhyML [35 (link)], using the HKY85 model [36 (link)].
Instead of weak DNA-DNA hybridization results for members of this genus [26 ] (data not shown) average nucleotide identity (ANI) was carried out according to the method described by Goris et al. [37 (link)] using the ezbiocloud platform (http://www.ezbiocloud.net/ezgenome/ani). According to Richter & Rosello-Mora [38 (link)] the cut-off for species boundary with this method is at 95–96%.

Blastp similarity searches were carried out on the open reading frames (ORFs) of both transcriptomes against the NCBI non-redundant (nr) database (downloaded 01 October 2013) with an e-value cut-off of 1e^-5 and a hit number threshold of 25. Blast2GO [140 (link)] with default parameters (hit adjusted to 25) was used for mapping and annotation of the ORFs, with InterProScan results merged with the already existing annotations. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations were also obtained via Blast2GO. To obtain information on the transcript expression pattern across different tissues, reads of the six individual tissues were mapped back to the non-redundant strand-specific and combined reference transcriptomes, using the CLC Genomics Workbench version 7.5 (CLC Bio, USA) with default parameters except for length and similarity fraction that were set to 1.0 and 0.9, respectively. General transcript patterns across the six tissues were determined, with the tissue distribution of specific transcripts determined based on the CLC mapping information and the results presented using Microsoft Excel graphs.

Exome enrichment was conducted following the manufacturer’s protocol for the ‘NimbleGen SeqCap EZ Human Exome v2.0’ beads (Roche NimbleGen Inc.). The kit interrogates a total of approximately 30,000 genes (~330,000 CCDS exons). Massively parallel sequencing was performed largely as described in Bentley et al. [20 (link)]. Whole exome capture and next-generation sequencing was carried out at Otogenetics Ltd. (www.otogenetics.com) on an Illumina HiSeq2000 (Illumina, San Diego, CA) platform and indexed libraries were subjected to paired-end (2×101 bp read length) sequencing-by-synthesis using fluorescent reversible terminators with a blocking group at the 3’-OH group. Three μg DNA of the affected mother E0023-I-2 was submitted for WES. Sequence reads were mapped to the human reference genome assembly (GRCh37/hg19) using CLC Genomics Workbench (version 7.5) software (CLC bio, Aarhus, Denmark). Variants were called, filtered, and prioritized according to their pathogenicity scores (>0.95) obtained from the Polyphen-2 web interface [21 (link)], MutationTaster [22 (link)], and CADD (>20) [23 (link)]. Furthermore, variants were cross-referenced with the Human Gene Mutation Database (HGMD, http://data.mch.mcgill.ca/phexdb), and genes known to be implicated in HR were intensively examined.