Sequencing the Genome of Indian Flying Fox, Natural Reservoir of Nipah Virus, Using Hybrid Assembly and Conservative Secondary Scaffolding.

Indian fruit bats, flying fox Pteropus medius was identified as an asymptomatic natural host of recently emerged Nipah virus, which is known to induce a severe infectious disease in humans. The absence of P. medius genome sequence presents an important obstacle for further studies of virus-host interactions and better understanding of mechanisms of zoonotic viral emergence. Generation of the high-quality genome sequence is often linked to a considerable effort associated to elevated costs. Although secondary scaffolding methods have reduced sequencing expenses, they imply the development of new tools for the integration of different data sources to achieve more reliable sequencing results. We initially sequenced the P. medius genome using the combination of Illumina paired-end and Nanopore sequencing, with a depth of 57.4x and 6.1x, respectively. Then, we introduced the novel scaff2link software to integrate multiple sources of information for secondary scaffolding, allowing to remove the association with discordant information among two sources. Different quality metrics were next produced to validate the benefits from secondary scaffolding. The P. medius genome, assembled by this method, has a length of 1,985 Mb and consists of 33,613 contigs and 16,113 scaffolds with an NG50 of 19 Mb. At least 22.5% of the assembled sequences is covered by interspersed repeats already described in other species and 19,823 coding genes are annotated. Phylogenetic analysis demonstrated the clustering of P. medius genome with two other Pteropus bat species, P. alecto and P. vampyrus, for which genome sequences are currently available. SARS-CoV entry receptor ACE2 sequence of P. medius was 82.7% identical with ACE2 of Rhinolophus sinicus bats, thought to be the natural host of SARS-CoV. Altogether, our results confirm that a lower depth of sequencing is enough to obtain a valuable genome sequence, using secondary scaffolding approaches and demonstrate the benefits of the scaff2link application. The genome sequence is now available to the scientific community to (i) proceed with further genomic analysis of P. medius, (ii) to characterize the underlying mechanism allowing Nipah virus maintenance and perpetuation in its bat host, and (iii) to monitor their evolutionary pathways toward a better understanding of bats' ability to control viral infections.

Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding.

The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbß3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet's ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis.

Whole-Genome Sequence of Mycobacterium abscessus Clinical Strain V06705.

Infection caused by Mycobacterium abscessus strains is a growing cause of concern in both community-acquired and health care-associated diseases, as these organisms naturally display multiple drug resistances. We report an annotated draft genome sequence of M. abscessus strain V06705 obtained from a patient in France.