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| Status |
Public on Sep 15, 2014 |
| Title |
Bp33 |
| Sample type |
SRA |
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| Source name |
Clinical
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| Organism |
Burkholderia pseudomallei |
| Characteristics |
strain: Bp33
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| Growth protocol |
Each Bp strain is grown on Tryptone-Soy Agar (Oxoid)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted using the QIAGEN Genomic Tip 500/G kit (QIAGEN, Valencia, CA), as per manufacturer’s instructions. Manipulations of live bacteria were performed in a BioSafety Level 3 facility in DSO National Laboratories.
To perform construction of a 17 kb SMRTbell™ library, genomic DNA (gDNA) was first analyzed on Field Inversion Gel Electrophoresis (FIGE) to ensure presence of high molecular weight gDNA. Twenty micrograms of gDNA was sheared using g-TUBEs (Covaris, Inc). Of the 20 ug sheared DNA, 10 ug were treated to repair apurinic/apyrimidinic sites, thymine dimers, nicks and gaps. Following this DNA damage repair, the ends of the double stranded fragments were polished to fill in any 5’ overhangs while removing any 3’ overhangs. The ends were then phosphorylated suitable for ligation with hairpin adapters supplied in the DNA Template Prep Kit 2.0 (3kb-10kb) (Pacific Biosciences, Menlo Park, CA. USA). The library was run on a BluePippin™ system (Sage Science, Inc., Beverly, MA., USA) to select for SMRTbell templates greater than 10 kb. The resulting average insert size was 17 kb based on 2100 Bioanalyzer (Agilent Technologies Genomics, Santa Clara, CA., USA). After size selection, sequencing primers were annealed to the hairpins of the SMRTbell templates followed by binding with the P4 sequencing polymerase (Pacific Biosciences, Menlo Park, CA. USA). To sequence the 17 kb SMRTbell library, the polymerase-bound templates were bound to MagBeads for zmw- loading in the PacBio RSII System at an on-plate concentration of 150 pM. They were subsequently sequenced using the DNA Sequencing Kit 2.0, with data collection of 180 mins (Pacific Biosciences, Menlo Park, CA. USA).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
PacBio RS II |
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| Description |
SMRTbell™ sequencing
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| Data processing |
PacBio Genome Assembly
Genomes were assembled using HGAP(Chin, Alexander, Marks, Klammer, Drake, Heiner, Clum, Copeland, Huddleston, Eichler et al. 2013) with default parameters in the SMRT Analysis Suite version 2.1 (Pacific Biosciences, Menlo Park, CA, USA). Additional manual assembly of contigs was carried out in cases of unique overlapping sequence. Consensus sequence polishing was done with the Quiver algorithm in GenomicConsensus version 0.7.0.
Base modification analysis
Base modification analysis was carried out by mapping SMRT sequencing reads to the respective assemblies using the BLASR mapper and the Pacific Biosciences' SMRT Analysis Suite version 2.1 using the standard mapping protocol. To identify modified positions, the pipeline uses kinetic signals acquired during SMRT sequencing compared to an in silico kinetic reference, and a t-test based kinetic score detection of modified base positions as described in http://www.pacb.com/pdf/TN_Detecting_DNA_Base_Modifications.pdf. The clustering of the sequence motifs was done using PacBio's Motif Finder (https://github.com/PacificBiosciences/DevNet/wiki/Motiffinder).
Genome_build: Not Applicable
Supplementary_files_format_and_content: comma separated value format and base modifications
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| Submission date |
Feb 19, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Tannistha Nandi |
| E-mail(s) |
[email protected]
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| Phone |
68088125
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| Organization name |
Genome Institute of Singapore
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| Department |
Cancer Therapeutics & Stratified Oncology
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| Street address |
60 Biopolis Street, Genome, #02-01
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| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
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| Platform ID |
GPL18301 |
| Series (1) |
| GSE55168 |
Methylome sequencing of Burkholderia pseudomallei strains Bp33 and Bp35 |
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| Relations |
| BioSample |
SAMN02645854 |
| SRA |
SRX475416 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1330706_Bp33_modifications.csv.gz |
199.0 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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