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No / Although the application of sequencing-by-synthesis techniques to DNA extracted from bones has revolutionized the study of ancient DNA, it has been plagued by large fractions of contaminating environmental DNA. The genetic analyses of hair shafts could be a solution: We present 10 previously unexamined Siberian mammoth (Mammuthus primigenius) mitochondrial genomes, sequenced with up to 48-fold coverage. The observed levels of damage-derived sequencing errors were lower than those observed in previously published frozen bone samples, even though one of the specimens was >50,000 14C years old and another had been stored for 200 years at room temperature. The method therefore sets the stage for molecular-genetic analysis of museum collections.

Yes / Aeromonas hydrophila is a well-known waterborne pathogen that recently was found to infect humans. Here, we report the draft genome of a freshwater isolate from a Malaysian waterfall, A. hydrophila strain M023, which portrays N-acylhomoserine lactone-dependent quorum sensing. / University of Malaya via High Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01, A-000001-50001, and UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

Yes / Pantoea stewartii strain M073a is a Gram-negative bacterium isolated from a tropical waterfall. This strain exhibits quorum-sensing activity. Here, the assembly and annotation of its genome are presented. / High Impact Research Grants from the University of Malaya (UM.C/625/1/HIR/MOHE/CHAN/01, grant no. A-000001-50001 and UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/ CHAN/14/1, no. H-50001-A000027)

Yes / Bacillus sp. is a Gram-positive bacterium that is commonly found in seawater. In this study, the genome of marine Bacillus sp. strain G3(2015) was sequenced using MiSeq. The fosfomycin resistant gene fosB was identified upon bacterial genome annotation. / University of Malaya through HIR grants (UM-MOHE HIR grant UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027; UM-MOHE HIR grant UM C/625/1/HIR/MOHE/CHAN/01, A000001- 50001); Postgraduate Research grant PG083-2015B

No / Background: Phylogenetic studies of bacteria have been based so far either on a single gene (usually the 16SrRNA) or on concatenated housekeeping genes. For what concerns the genus Mycobacterium these approaches support the separation of rapidly and slowly growing species and the clustering of most species in well-defined phylogenetic groups. The advent of high-throughput shotgun sequencing leads us to revise conventional tax-onomy of mycobacteria on the light of genomic data. For this purpose we investigated 88 newly sequenced species in addition to 60 retrieved from GenBank and used the Average Nucleotide Identity pairwise scores to reconstruct phylogenetic relationships within this genus.Results:Our analysis confirmed the separation of slow and rapid growers and the intermediate position occupied by the M. terrae complex. Among the rapid growers, the species of the M. chelonae-abscessus complex belonged to the most ancestral cluster. Other major clades of rapid growers included the species related to M. fortuitum and M. smegmatis and a large grouping containing mostly environmental species rarely isolated from humans. The members of the M. terrae complex appeared as the most ancestral slow growers. Among slow growers two deep branches led to the clusters of species related to M. celatum and M. xenopi and to a large group harboring most of the species more frequently responsible of disease in humans, including the major pathogenic mycobacteria (M.tuberculosis,M. leprae,M. ulcerans). The species previously grouped in the M. simiae complex were allocated in a number of sub-clades; of them, only the one including the species M. simiae identified the real members of this complex. The other clades included also species previously not considered related to M. simiae. The ANI analysis,in most cases supported by Genome to Genome Distance and by Genomic Signature-Delta Difference, showed that a number of species with standing in literature were indeed synonymous.Conclusions:Genomic data revealed to be much more informative in comparison with phenotype. We believe that the genomic revolution enabled by high-throughput shotgun sequencing should now be considered in order to revise the conservative approaches still informing taxonomic sciences.

Yes / Understanding the circulating Mycobacterium tuberculosis resistance mutations is vital for better TB control strategies, especially to inform a new MDR-TB treatment programme. We complemented the phenotypic drug susceptibility testing (DST) based drug resistance surveys (DRSs) conducted in Uganda between 2008 and 2011 with Whole Genome Sequencing (WGS) of 90 Mycobacterium tuberculosis isolates phenotypically resistant to rifampicin and/or isoniazid to better understand the extent of drug resistance. A total of 31 (34.4 %) patients had MDR-TB, 5 (5.6 %) mono-rifampicin resistance and 54 (60.0 %) mono-isoniazid resistance by phenotypic DST. Pyrazinamide resistance mutations were identified in 32.3% of the MDR-TB patients. Resistance to injectable agents was detected in 4/90 (4.4%), and none to fluoroquinolones or novel drugs. Compensatory mutations in rpoC were identified in two patients. The sensitivity and specificity of drug resistance mutations compared to phenotypic DST were for rpoB 88.6% and 98.1%, katG 60.0% and 100%, fabG1 16.5% and 100%, katG and/or fabG1 71.8% and 100%, embCAB 63.0% and 82.5%, rrs 11.4% and 100%, rpsL 20.5% and 95.7% and rrs and/or rpsL 31.8% and 95.7%. Phylogenetic analysis showed dispersed MDR-TB isolate, with only one cluster of three Beijing family from South West Uganda. Among tuberculosis patients in Uganda, resistance beyond first-line drugs as well as compensatory mutations remain low, and MDR-TB isolates did not arise from a dominant clone. Our findings show the potential use of sequencing for complementing DRSs or surveillance in this setting, with good specificity compared to phenotypic DST. The reported high confidence mutations can be included in molecular assays, and population-based studies can track transmission of MDR-TB including the Beijing family strains in the South West of the country. / Erasmus Mundus Joint Doctorate Program of the European Union through a training grant to WS and the European Research Council-INTERRUPTB starting grant (nr.311725) to BdJ.

Yes / Whole genome sequencing (WGS) is increasingly used for Mycobacterium tuberculosis (Mtb) research. Countries with the highest tuberculosis (TB) burden face important challenges to integrate WGS into surveillance and research. We assessed the global status of Mtb WGS and developed a 3-week training course coupled with long-term mentoring and WGS infrastructure building. Training focused on genome sequencing, bioinformatics and development of a locally relevant WGS research project. The aim of the long-term mentoring was to support trainees in project implementation and funding acquisition. The focus of WGS infrastructure building was on the DNA extraction process and bioinformatics. Compared to their TB burden, Asia and Africa are grossly underrepresented in Mtb WGS research. Challenges faced resulted in adaptations to the training, mentoring and infrastructure building. Out-of-date laptop hardware and operating systems were overcome by using online tools and a Galaxy WGS analysis pipeline. A case studies approach created a safe atmosphere for students to formulate and defend opinions. Because quality DNA extraction is paramount for WGS, a biosafety level 3 and general laboratory skill training session were added, use of commercial DNA extraction kits was introduced and a 2-week training in a highly equipped laboratory was combined with a 1-week training in the local setting. By developing and sharing the components of and experiences with a sequencing and bioinformatics training program, we hope to stimulate capacity building programs for Mtb WGS and empower high-burden countries to play an important role in WGS-based TB surveillance and research. / Vlaamse Interuniversitaire Raad-secretariaat voor universitaire ontwikkelingssamenwerking (ET2018JOI008A10); the Research Foundation Flanders under FWO Odysseus (grant G0F8316N); the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation of South Africa (64751); the South African Medical Research Council.

no / The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.