If they shared the same mapping position, stemmed from the same
If they shared the same mapping position, stemmed from the same DNA strand and possessed exactly the PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28461585 same sequence. In the case of duplicates, the read having the best quality sum was preserved. The resulting alignment maps were then analysed by using SNIFER (https://bitbucket.org/clafooty/tango/wiki/Home) for the SNP calling, which is based on a comparison of aligned read sequences to the reference genome from mapping positions. Mismatches detected were then filtered according to 5 stringent criteria: (i) a coverage sum > 10; (ii) a substitution frequency of at least 0.89; (iii) a mean quality of mapped bases > 20 according to the Sanger format; and both mean (iv) coverage and (v) quality >20 for the 10 bases surrounding the variant (-5/+5). So as to investigate large insertion-deletion events, each short-read data set was de novo assembled using the perl script VelvetOptimiser, provided with the VELVET package [72].Based on the results obtained from the Bayesian algorithm STRUCTURE, three “hybrid” Peficitinib web strains from the MAB were selected for whole-genome sequencing (Strain M139, strain 23 and strain 137). Strain M139 came from a sputum sample from a Malaysian patient with a MAB lung infection; contigs were already generated and assembled in a former study [50]. Strains 137 and 23 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28388412 (respectively Mycobacterium abscessus subsp. bolletii 137 and Mycobacterium abscessus 23) were taken from our laboratory collection. Libraries were constructed using the Nextera Kit (Illumina) from 50 ng of DNA according to Illumina’s recommendations. Pooled libraries were sequenced on an Illumina HiSeq-2000 platform to generate 100 bp paired reads, with the TruSeq PE Cluster kit v3 and TruSeq SBS kit v3 (Illumina). All reads were pre-processed to remove low quality or artefactual nucleotides. First, all nucleotides occurring at 5 and 3 ends and supported by a Phred quality score < 28 were trimmed off using SICKLE (https://github.com/najoshi/sickle). Second, contaminant oligonucleotides (i.e., library adaptors) were detected and trimmed off using ALIENTRIMMER [73]. Third, reads shorter than 45 nt after the aforementioned cleaning steps were discarded, as were those containing more than 5 nucleotides with Phred score < 28. Finally, the program FQDUPLICATE (ftp://ftp.pasteur.fr/pub/gensoft/projects/ fqtools) was used to discard every duplicate single- or paired-ends reads. A de novo assembly of the remaining reads was built with CLC Genomics Workbench version 3 (CLC Bio, Cambridge, MA). Contigs were then reordered using the MUMMER software. The contigs of the three "hybrid" strains were compared with the three reference strains previously assembled (M. abscessus subspecies, M. massiliense subspecies, and M. bolletii subspecies) using NUCMER [74] and the deltafilter command (90 minimum identity threshold on at least 400 nucleotides). The Show-Tilling script was then used to determine the order of the contigs (minimum 10 coverage, maximal gap length 100,000 bp). The rejected contigs were manually checked and reintegrated in the final assembly if they had at least 96 identity on > 1500 bp regions. The assembled genomes of strains M139, 23 and 137 are very similar to the reference strains (4,916,028 bp, 4,834,006 bp, and 5,011,043 bp, respectively), and represent at least 95 of the size of the longest reference M. abscessus subsp. abscessus strain chromosome, that was sequenced by the Sanger method [41].Sapriel et al. BMC Genomics (2016) 17:Page 14 ofMycobacter.
