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| Status |
Public on Jul 16, 2014 |
| Title |
WT8 |
| Sample type |
SRA |
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| Source name |
Blood leukocytes, PD, post-DBS, ON-Stim
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| Organism |
Homo sapiens |
| Characteristics |
disease status: Parkinson's Disease (PD)
gender: male
cell type: blood leukocytes
treatment: post-deep brain stimulation (DBS) on electrical stimulation
control volunteer/patient number: PD2
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| Growth protocol |
Leukocytes were filtered from total blood samples (collected with EDTA vaccutainer tubes) with Ambion Inc. LeukoLOCK™ filters within up to 10 minutes of blood extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The captured leukocytes were treated with RNAlater immediately after filtration of the blood and were maintained frozen until RNA extraction.
RNA quality was assessed by running the samples on Agilent RNA 6000 Nano gels (#5067-1511). For each library, ribosomal RNA of 5 ug total RNA was removed using the Invitrogen RiboMinus kit (# A10837-08), and then the sample was concentrated using the RiboMinus™ Concentration Module (Invitrogen). Ribosomal RNA removal was verified by RNA 6000 Nano gel analysis. Library construction was done according to the SOLiD™ Whole Transcriptome Analysis Kit (PN 4425680) protocol. Fragmentation (by RNase III) was verified on Agilent RNA 6000 Pico Kit (#5067-1513), and 150 ng fragmented RNA were used for further protocol. cDNA samples were run on 4% agarose gel, 150-250bp fragments were cut and extracted using the Qiagen Min-Elute Gel Extraction Kit (#28604), and the gel was dissolved by intensive vortexing and not by heating. Libraries were barcoded and amplified for 12 cycles using barcoded primers supplied in the SOLiD™ Transcriptome Multiplexing Kit (Ambion 4427046). Libraries were quantified using the Kapa ABI SOLiD Library Quantification Kit (KK4833) and diluted for final analysis on Agilent High Sensitivity DNA Kit (#5067-4626). 500 uM libraries were used for emulsion PCR according to the Applied Biosystems SOLiD™ 3 System Templated Bead Preparation Guide (4407421 ) to prepare for sequencing on the SOLiD 3 platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD System 3.0 |
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| Description |
Parkinson's Disease (PD) patient, post-DBS on electrical stimulation (ON-Stim).
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| Data processing |
Primary data analysis: SOLiD 3 System software analysis was used for primary data analysis including image analysis, bead finding, quality metrices and color calls. The software applications used to set and control data analysis included the SOLiD software suite under license agreement. The suite included: Instrument Control Software (ICS), SOLiD Experimental Tracking System (SETS), and SOLiD Analysis Tools (SAT) V3.0. The jobs were managed by the Job Manager. The Corona-Lite v4.0 platform was used. Sequencing was run on the Applied Biosystems SOLiD 3 System to generate sequencing data. Images of each cycle were analyzed, data was clustered and normalized. For each tag, a sequential (sequence-ordered) set of color space calls was produced. Quality metrices were produced through normalization. Two probe sets were used to maximize the fraction of "mappable" amplified beads, read length and sequencing throughput for sequencing of the 50-bp reads. Five rounds of primers (A, B, C, D and E) were used to sequence template by ligation of di-base labeled probes. As the libraries were size fragmented, the set of primers used was specific to the P1 Adaptor. For each library, three types of raw data files were created: .csfasta (the sequenced reads in color space), .qual and .stats. The quality values given in the .qual files are estimate of confidences given for each color call. The quality value q for a particular xcall is mathematically related to its probability of error p. q=-10log10p. The SOLiD QVs are similar to those generated by Phred and the KB basecaller for capillary electrophoresis (references: I. Ewing B., green p., 1998, Base-calling of automated sequencer traces using Phred. II. Error probabilities: Genome Research 8:186-194). The algorithm relies on training (calibration) to a large set of control data and color calls for which the correct call is known. In the SOLiD 3 system, the correct call is determined by mapping the read to a known reference sequence.
Color space conversion: Through color space analysis, sequential ligation of dye-labeled oligonucleotides enabled massively parallel sequencing of clonally amplified DNA fragments through the SOLiD 3 system. Each color represented 4 potential two-base combinations. The conversion into nucleotide base space was conducted during the secondary analysis data phase.
Secondary data analysis: The SOLID secondary data analysis included matching of the reads to the reference genome and generation of base space sequences. The .gff and .sam files were created during this analysis step. The reads were mapped to the human genome version homo_sapiens.GRCh37.56.dna.toplevel.fa database. The alignment software Mapreads was used. Count merging was performed in both pipelines. It employs discontiguous word pattern search algorithms.
Tertiary data analysis: Exon- and junction-level mapping was conducted using Applied BioScope V1.2 software through cloud computing. The analysis was conducted on the ordered genome. Junction mapping was conducted using the splice junction extractor tool. Exon-level mapping was conducted using the counttag BioScope tool.
Genome_build: GRCh37
Supplementary_files_format_and_content: .gff, .bed, and .tab files were created using BioScope.
Supplementary_files_format_and_content: GFF (General Feature Format) is a record-based file format where each line describes a single feature (i.e., read) with a list of tab-delimited fields in a fixed order specified by the GFF specifications. The file type created by the SOLiD 3 system is the .gff v3 file.
Supplementary_files_format_and_content: The .bed files are tab-delimited text files that define a feature track.
Supplementary_files_format_and_content: For each library, .tab files were generated for junctions. The file includes the following fields: Exon-1 (the gene id followed by the exon order on the gene), Exon-1-reference, Exon-1 strand (+/-), Exon-1 start position, Exon-1 end position, Exon-2, Exon-2-reference, Exon-2 strand, Exon-2 start, Exon-2 end, Exon-1-size, Exon-2-size, Exon-1-readcount, Exon-2-readcount, Exon-1-F3-RPKM, Exon-2-F3-RPKM, Exon-distance (distance between two exons; the exon distance is not applicable if it is on a different chromosome), Total-SR-evidence (the total single-read evidence for the junction), Unique-SR-evidence, JCV (a Junction confidence value), Known ("K" if a junction is known, and "P" if a junction is putative), E1-all-genes (the list of all genes to which exon-1 was mapped), E2-all-genes (the list of all genes to which exon-1 was mapped), Other (other information provided about the junction).
Supplementary_files_format_and_content: RPKM in the .tab files is computed by 10^9x(ExonReadCount/TotalReadCount ExonLength).
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| Submission date |
Jun 25, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Lilach Soreq |
| E-mail(s) |
l.soreq@ucl.ac.uk
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| Phone |
+44 07938689793
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| Organization name |
UCL
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| Department |
Molecular Neuroscience
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| Lab |
Prof. John Hardy
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| Street address |
Queen Square
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| City |
London |
| ZIP/Postal code |
WC1N 3BG |
| Country |
United Kingdom |
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| Platform ID |
GPL9442 |
| Series (1) |
| GSE42608 |
Whole-transcriptome high-throughput RNA sequencing of blood leukocytes from Parkinson's disease human patients pre- and post-deep brain stimulation |
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| Relations |
| BioSample |
SAMN02213618 |
| SRA |
SRX315053 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1174037_solid0162_20100916_WT8_F3.csfasta.ma.50.6.annotated.gff3.contig.range.txt.gz |
322 b |
(ftp)(http) |
TXT |
| GSM1174037_solid0162_20100916_WT8_F3.csfasta.ma.50.6.annotated.gff3.gz |
160.3 Mb |
(ftp)(http) |
GFF3 |
| GSM1174037_wt8.sr.junctions.bed.gz |
1.8 Mb |
(ftp)(http) |
BED |
| GSM1174037_wt8.sr.junctions.tab.gz |
1.5 Mb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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