Characterization of the regions surrounding zooA-zif and lss-lif

Staphylococcus simulans biovar staphylolyticus produces the staphylolytic enzyme lysostaphin and it is the only known lysostaphin producer within the species. Genes with similarity to the lysostaphin gene (lss) and the gene for lysostaphin resistance (lif) have been characterized, including the chromosomally encoded streptococcolytic enzyme zoocin A (zooA) and its immunity factor (zif) from Streptococcus equi subsp. zooepidemicus strain 4881. Both zooA - zif and lss - lif are divergently transcribed and the similarity in genetic arrangement between these loci generated the hypothesis that zif and zooA were acquired by horizontal gene transfer. Initially, twenty - four S. equi subsp. zooepidemicus strains were analyzed to determine the prevalence and acquisition of the zooA - zif locus within the subspecies. While zooA - zif is not common within the subspecies, PFGE and RAPD showed that the 24 strains were genetically diverse. Sequences derived from strain 4881 revealed that zooA - zif are flanked by two transposon like sequences and are integrated into the chromosome adjacent to the gene flaR. A comparison of the flaR region of S. equi subsp. zooepidemicus strains 4881, 9g, H70 and MGCS10565, as well as the related S. equi subsp. equi strain 4047, suggested that flaR marks a region of genome plasticity in these streptococci. S. simulans bv. staphylolyticus contains five plasmids designated pACK1 - pACK5. lss and lif are encoded on the largest plasmid, pACK1. In order to investigate the regions flanking lss and lif, as well as to understand the relationship between pACK1 and pACK3 within the strain, the sequences of pACK1 (55171 bp) and pACK3 (28613 bp) were determined. Comparison of the two plasmids revealed that they are virtually identical within an approximately 28 kb common region. Sequences flanking the common region contain IS431 elements and direct repeats mark where the two plasmid sequences diverge, providing evidence that pACK1 was derived from pACK3 by insertion of sequences unique to pACK1 into pACK3. lss and lif are located within the region unique to pACK1 and, as with zooA - zif, are flanked by two transposon like sequences. The presence of flanking transposons near zooA - zif and lss - lif suggests that these organisms may have received these genes by horizontal gene transfer. (Published By University of Alabama Libraries

Optimization of Next-Generation Sequencing Informatics Pipelines for Clinical Laboratory Practice

We direct your readers’ attention to the principles and guidelines (see Supplementary Guidelines) developed by the Next-generation Sequencing: Standardization of Clinical Testing II (Nex-StoCT II) informatics workgroup, which was convened by the Centers for Disease Control and Prevention (CDC). This work represents the first effort to systematically review current practices and present consensus recommendations for the design, optimization, and implementation of an informatics pipeline for clinical next-generation sequencing (NGS) in compliance with existing regulatory and professional quality standards1. Workgroup participants included informatics experts, clinical and research laboratory professionals, physicians with experience in NGS results interpretation, NGS test platform and software developers, and participants from US government agencies and professional organizations. The primary focus was the design, optimization, and implementation of an NGS informatics pipeline for the detection of germline sequence variants; however, the workgroup also discussed use of NGS for cancer and infectious disease testing. The typical NGS analytical process and selected workgroup recommendations are summarized in Table 1, Supplementary Fig. 1 and the Supplementary Guidelines

A survey of current practices for genomic sequencing test interpretation and reporting processes in US laboratories

PURPOSE While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation and reporting of clinically significant variants. This study aimed to examine workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization. METHODS Surveys and follow-up interviews were conducted with laboratories offering exome and/or genome sequencing, to support a research program or for routine clinical services. The 73-item survey elicited multiple choice and free text responses, later clarified with phone interviews. RESULTS Twenty-one laboratories participated. Practices highly concordant across all groups included: consent documentation, multi-person case review, and enabling patient opt-out of incidental or secondary findings analysis. Noted divergence included use of phenotypic data to inform case analysis and interpretation, and reporting of case-specific quality metrics and methods. Few laboratory policies detailed procedures for data reanalysis, data sharing or patient access to data. CONCLUSION This study provides an overview of practices and policies of experienced exome and genome sequencing laboratories. The results enable broader consideration of which practices are becoming standard approaches, where divergence remains, and areas development of best practice guidelines may be helpful