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| Status |
Public on Sep 25, 2019 |
| Title |
Bp D286 |
| Sample type |
SRA |
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| Source name |
Clinical isolate
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| Organism |
Burkholderia pseudomallei |
| Characteristics |
strain: D286
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| Growth protocol |
The isolates were grown overnight at 37°C in Brain Heart Infusion broth (BHIB) and plated on Ashdown selective agar plates.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The genomic DNA for each isolate was extracted using modifications to the standard protocol described by (Sambrook and Russell, 2001). Briefly, the isolates were grown overnight at 37°C in Brain Heart Infusion broth (BHIB) and plated on Ashdown selective agar plates. A single colony was inoculated into BHIB and grown overnight at 37°C. Pellets from the overnight cultures were resuspended in the remaining 50 μL of supernatant with additional 100ug/ml proteinase K and incubated at 42°C for 10 minutes and 56°C for 30 minutes. Samples were cooled to room temperature and 5 µg RNase A were added to the samples, mixed and incubated for 30 minutes at 37°C. DNA was purified using phenol-chloroform-isoamyl alcohol (25:24:1 ratio), precipitated with ethanol, spooled into deionized water and stored at -20°C.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
PacBio RS II |
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| Description |
Submitter states that efforts to obtain raw data files were unsuccessful
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| Data processing |
Genome sequencing and assembly: The PacBio sequencing reads were filtered and assembled using RS_HGAP_Assembly protocol in SMRT Portal v2.1.1. This involved one round of quiver correction to improve assembly and errors corrections. Three rounds of quiver corrections were then carried out using the RS_Resequencing.1 protocol to obtain very high consensus accuracy (99.998%). After the preliminary assemblies were obtained, the start sites of the assembled genomes were determined based on the reference start site while both 5' and 3' ends were connected by inserting 100 Ns between the cut sequences. This was followed by 4 rounds of quiver corrections to improve the assembly accuracy to 99.999% and close the final 100 Ns gap region.
Gene prediction and genome annotation: Gene predictions for the B. pseudomallei genomes were performed using NCBI's Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al., 2016). PGAP pipeline uses tBLASTn together with ProSplign to detect frameshifted genes and pseudogenes, and subsequently uses GeneMarkS+ program to identify protein coding regions. The annotation of proteins’ functions were performed by BLASTp searches against NCBI Protein Clusters Database (ProtClustDB) (Klimke et al., 2009), UniProt-SwissProt Protein Knowledgebase (Uniprot Consortium, 2015) and curated bacteriophage proteins in RefSeq database.
Base modification Analysis and Motif Detection: Methylation analysis was carried out by RS_Modification_and_Motif_Analysis.1 protocol in SMRT Portal. Raw reads were filtered using SFilter in order to remove short reads and adapter before the high quality reads were aligned using the BLASTR mapper (Bussotti et al., 2011). The base modifications were detected via analysis of the base addition kinetics data where significant changes in the interpulse duration (IPD) indicate the presence of either m6A, m4C or m5C modified sites (Flusberg et al., 2010). The motifs associated with the modified bases were identified using MotifFinder and based on the modification quality value (QV) score, they were then determined to be either methylated or unmethylated
Submitter states that efforts to obtain raw data files were unsuccessful
Supplementary_files_format_and_content: Comma separated value format and base modifications in gff format
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| Submission date |
Sep 24, 2019 |
| Last update date |
Sep 25, 2019 |
| Contact name |
Hirzahida Mohd Padil |
| E-mail(s) |
eda.pixie@gmail.com, hirzahida@mfrlab.org
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| Organization name |
Universiti Kebangsaan Malaysia
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| Street address |
Bangi
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| City |
Kajang |
| State/province |
Selangor |
| ZIP/Postal code |
43600 |
| Country |
Malaysia |
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| Platform ID |
GPL18301 |
| Series (1) |
| GSE137930 |
Methylome sequencing of Burkholderia pseudomallei D286, H10, PMC2000, R15 and 982 |
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| Relations |
| BioSample |
SAMN12830277 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4094591_Bp_D286_methylome.txt.gz |
211.5 Mb |
(ftp)(http) |
TXT |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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