Project description:Whole-genome sequencing is an important way to understand the genetic information, gene function, biological characteristics, and living mechanisms of organisms. There is no difficulty to have mega-level genomes sequenced at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. The shotgun sequencing method failed to dissect this genome. After insisting for 10 years and going over 3 generations of sequencing techniques, we successfully dissected the PaP1 genome with 91,715 bp in length. Single-molecule sequencing revealed that this genome contains lots of modified bases, including 51 N6-methyladenines (m6A) and 152 N4-methylcytosines (m4C). At the same time, further investigations revealed a novel immune mechanism of bacteria, by which the host bacteria can recognize and repel the modified bases containing inserts in large scale, and this led to the failure of the shotgun method in PaP1 genome sequencing. Strategy of resolving this problem is use of non-library dependent sequencing techniques or use of the nfi- mutant of E. coli DH5M-NM-1 as the host bacteria to construct the shotgun library. In conclusion, we unlock the mystery of phage PaP1 genome hard to be sequenced, and discover a new mechanism of bacterial immunity in present study. Methylation profiling of Pseudomonas aeruginosa phage PaP1 using kinetic data generated by single-molecule, real-time (SMRT) sequencing on the PacBio RS.
Project description:Endometriosis is a chronic, estrogen-dependent gynecological condition that affects approximately 10% of women of reproductive age. The most widely accepted theory of the etiology of endometriosis includes the process of retrograde menstruation, where menstrual effluent travels up the Fallopian tubes, accesses the peritoneal cavity, and in some people is able to establish endometriotic lesions. Recent reports suggest the uterus is not devoid of bacteria, as was once believed. Thus, the refluxed menstrual effluent may also carry bacteria along with it, and this bacteria has been suggested to contribute to inflammation, and establishment and growth of endometriotic lesions. Here, we sought to compare and contrast the uterine bacteria (endometrial microbiota) in women with surgically confirmed presence or absence of endometriosis using next-generation 16S rRNA gene sequencing. We obtained an average of more than 9000 sequence reads per endometrial biopsy, and found that the endometrial microbiota of women with endometriosis was more diverse (greater Shannon Diversity Index and greater proportion of ‘Other’ taxa) than that of symptomatic controls (women with pelvic pain, but with surgically confirmed absence of endometriosis; diagnosed with other benign gynecological conditions at surgery). The difference in endometrial microbiotas was supported in unsupervised cluster analyses where some clustering of endometrial microbiota by disease status (endometriosis vs. controls) was observed. The bacterial taxa enriched in the endometrial microbiota of women with endometriosis belonged to the Actinobacteria phylum, Oxalobacteraceae and Streptococcaceae families, and Tepidimonas genus, while those enriched in the symptomatic controls (without endometriosis) belonged to the Burkholderiaceae family, and Ralstonia genus. Taken together, our findings suggest the endometrial microbiota is perturbed in people with endometriosis.