Aims : DNA sequencing is a powerful tool and less time-consuming for bacterial detection and identification. The aim of this study was to compare the application of the Oxford Nanopore MinION™ sequencing device for direct DNA sequencing from clinical specimens with the routine workup. Methodology and results : We used conventional bacteriological-based methods to detect and identify bacterial pathogens in 10 clinical specimens. In addition, the 16S metagenomic sequencing was performed by using a MinION™sequencing device with barcoded primers of a 16S Barcoding kit (Code N° SQK-RAB204, Oxford Nanopore Technologies, UK). The DNA was amplified by PCR using specific 16S primers (27F and 1492R) that contain barcodes and 5' tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters of the 16S Barcoding kit. Data wasanalyzed with WIMP and EPI2ME to classify and identify species in real-time. Ten clinical specimens were processed for bacterial isolation. A total of 8 urine samples were subjected to culture-dependent methods, successfully identifying the presence of pathogenic bacteria. Out of the total eight urine samples, both methods successfully identified six bacterial pathogens. Escherichia coli were identified, and the others were detected as Salmonella enterica, Veillonella parvula and Streptococcus anginosus using MinION™ sequencing. Two urine samples had different results. Escherichia coli was detected directly through MinION™ sequencing, bypassing the need for culture results. Conclusion, significance and impact of study MinION™ sequencing of 16S rRNA genes could accurately detect diverse bacterial pathogens in clinical specimens. Additionally, the bacterial species classification generated by analyzing 16S rRNA gene sequences can be helpful for rapid identification. The whole procedure takes less than 8 h to complete; same-day diagnosis can be completed.

PURPOSE: Germline mutations within melanoma susceptibility genes are present only in minority of melanoma patients and it is expected that additional genes will be discovered with next generation sequence technology and whole-exome sequencing (WES). MATERIALS AND METHODS: Herein we performed WES on a cohort of 96 unrelated Polish patients with melanoma diagnosed under the age of 40 years who all screened negative for the presence of CDKN2A variants. A replication study using a set of 1,200 melanoma patient DNA samples and similarly large series of healthy controls was undertaken. RESULTS: We selected 21 potentially deleterious variants in 20 genes (VRK1, MYCT1, DNAH14, CASC3, MS4A12, PRC1, WWOX, CARD6, EXO5, CASC3, CASP8AP2, STK33, SAMD11, CNDP2, CPNE1, EFCAB6, CABLES1, LEKR1, NUDT17, and RRP15), which were identified by WES and confirmed by Sanger sequencing for an association study. Evaluation of the allele distribution among carriers and their relatives in available family trios revealed that these variants were unlikely to account for many familial cases of melanoma. Replication study revealed no statistically significant differences between cases and controls. CONCLUSION: Although most of the changes seemed to be neutral we could not exclude an association between variants in VRK1, CREB3L3, EXO5, and STK33 with melanoma risk.
Alleles ; Cohort Studies ; DNA ; Exome* ; Frameshift Mutation ; Germ-Line Mutation ; Humans ; Incidence* ; Melanoma* ; Poland*