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SRX337967: GSM1214207: PaP1; Pseudomonas phage PaP1; OTHER
2 PACBIO_SMRT (PacBio RS) runs: 17,380 spots, 18M bases, 16.7Mb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: Unlock the mystery of the hard-to-sequence phage genome: discovering a novel mechanism of bacterial immunity
show Abstracthide Abstract
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 DH5a 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. Overall design: Methylation profiling of Pseudomonas aeruginosa phage PaP1 using kinetic data generated by single-molecule, real-time (SMRT) sequencing on the PacBio RS.
Sample: PaP1
SAMN02324540 • SRS472358 • All experiments • All runs
Library:
Instrument: PacBio RS
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: EDTA (20 mM), proteinase K (50 μg mL-1), and sodium dodecyl sulfate (0.5%, w/v) were added to the purified phage stock solution (PaP1 or PaP3). The mixture was incubated at 56 °C for 1 h, then an equal volume of phenol–chloroform–isoamyl alcohol solution (25:24:1) was added, followed by centrifugation at 5,000 g for 10 min. The aqueous layer was collected and extracted with chloroform at 5,000 g for 10 min. Finally, the collected aqueous layer was mixed with 0.6 volumes of isopropanol, and stored overnight at –20 °C. The next day, the mixture was centrifuged for 10 min at 12,000 g and 4 °C, and the precipitated DNA was collected and washed with 70% and 100% ethanol, respectively. The PaP1 DNA was suspended in TE buffer (pH 8.0) and stored at –20 °C for subsequent use. Genomic DNA was randomly sheared to an avearge size of approximately 2,000 bp, end repaired, and ligated to blunt end hairpin adapters for form standard SMRTbell templates
Experiment attributes:
GEO Accession: GSM1214207
Links:
External link:
Runs: 2 runs, 17,380 spots, 18M bases, 16.7Mb
Run# of Spots# of BasesSizePublished
SRR9554639,0859.4M8.7Mb2013-12-02
SRR9554648,2958.6M8Mb2013-12-02

ID:
475510

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