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Sample GSM2155607 Query DataSets for GSM2155607
Status Public on Mar 12, 2018
Title T50
Sample type SRA
 
Source name Primary breast tumor
Organism Homo sapiens
Characteristics tissue: breast tumor
er ihc routinestain clinical reading: 0
er ihc routinestain reading 2: 0
er ihc routinestain reading 3: 0
er ihc restain reading 1: 0
er ihc restain reading 2: 0
er ihc restain reading 3: 0
er consensus: 0
pgr ihc routinestain clinical reading: 0
pgr routinestain reading 2: 0
pgr routinestain reading 3: 0
pgr restain reading 1: 0
pgr restain reading 2: 0
pgr restain reading 3: 0
pgr consensus: 0
her2 ihc clinreading: 3
her2 ihc reading 2: 2
her2 ihc reading 3: 3
her2 clinical status: 1
her2 sish reading 1: 4
her2 sish reading 2: 4
her2 sish reading 3: 4
her2 consensus: 1
ki67 reading 1: 30
ki67 reading 2: 30
ki67 reading 3: 60
ki67 consensus: 30
nhg reading 1 tubular: 3
nhg reading 1 pleomorph: 3
nhg reading 1 mitotic: 3
nhg reading 2 tubular: 3
nhg reading 2 pleomorph: 3
nhg reading 2 mitotic: 3
nhg reading 3 tubular: 3
nhg reading 3 pleomorph: 3
nhg reading 3 mitotic: 2
nhg consensus: 3
pam50 subtype: Her2
Extracted molecule polyA RNA
Extraction protocol QIAGEN AllPrep
Poly(A) mRNA is isolated from the total RNA in up to 96-well microtiter plate format by two rounds of purification with Dynabeads Oligo (dT)25 (Invitrogen) using a KingFisher Flex magnetic particle processor (ThermoScientific). Zinc-mediated fragmentation (Ambion) is performed and the fragmented mRNA retrieved using column purification (Zymo-Spin I-96 plates; Zymo). The sequencing library generation protocol is a modification of the dUTP method, which importantly retains the directionality (stranded-ness) of the sequenced RNA molecules. First strand cDNA synthesis is performed using random hexamers and standard dNTP mix followed by cleanup using Sephadex gel filtration (Illustra AutoScreen-96A plates; GE Healthcare), and second strand cDNA synthesis is performed using dUTP in place of dTTP in the dNTP-mix and cleanup using Zymo-Spin I-96 plates. The cDNA is end-repaired and A-tailed, and diluted TruSeq adapters with barcodes are ligated using a modified protocol (Illumina). Adapter-ligated cDNA is then size-selected to remove short oligonucleotides using carboxylic acid (CA) paramagnetic beads (Invitrogen) and polyethylene glycol (PEG), similar to the previously described methods, and automated on the KingFisher Flex. The second cDNA strand is digested using uracil-DNA glycosylase and the product is enriched by 12 PCR cycles (Illumina). The PCR product undergoes two cycles of size selection using CA-beads and varying concentrations of PEG, first to exclude DNA fragments >700 bp and then to exclude fragments <200 bp. Quality control is performed on control libraries using Qubit fluorometric measurement (Life Technologies) and Caliper LabChip XT microcapillary gel electrophoresis. Typically, 10-24 barcoded libraries are included in a pool and each pool is sequenced in at least one lane across dual flowcells. Paired-end sequencing of 50 bp read-length is performed on an Illumina HiSeq 2000 instrument.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Description For pathology key, see pathology_consensus_key.xlsx
Data processing Base-calling using manufacturer's on-instrument software.
Demultiplexing using the Picard ExtractIlluminaBarcodes algorithm.
Filtering to remove reads that align (using Bowtie 2 with default parameters except -k 1 --phred33 --local) to ribosomal RNA/DNA (GenBank loci NR_023363.1, NR_003285.2, NR_003286.2, NR_003287.2, X12811.1, U13369.1), phiX174 Illumina control (NC_001422.1), and sequences contained in the UCSC hg19 RepeatMasker track (downloaded March 14, 2011).
Remaining reads are aligned using TopHat2 2.0.5 (default parameters except for --mate-inner-dist (average size with adapters 355, range 268-465, measured for each sample individually) --mate-std-dev 100 --library-type fr-firststrand --keep-fasta-order --no-coverage-search) to the human genome reference GRCh37/hg19 (with b37 masked chromosome Y and hs37d5 decoy sequences) together with 80,883 transcript annotations from the UCSC knownGenes table (downloaded September 10, 2012).
Gene expression data in FPKM were generated using cufflinks 2.1.1 and pre-processed by collapsing on 27,979 unique gene symbols (sum of FPKM values of each matching transcript), keeping 18,802 NCBI RefSeq NM-category (mRNA) gene symbols and adding to each expression measurement 0.1 FPKM, performing a log2 transformation.
PAM50 subtyping was performed using an implementation of the Parker method (Parker et al., J.Clin Oncol 2009). In short, to avoid context dependency when assigning PAM50 subtype by nearest-centroid, a fixed reference was selected to match the original cohort used by Parker et al. with respect to available clinical characteristics. Before subtyping tumors in this study, gene expression of the PAM50 genes for each tumor was centered to the reference set separately using custom R scripts.
Genome_build: Human genome reference GRCh37/hg19 (with b37 masked chromosome Y and hs37d5 decoy sequences).
Supplementary_files_format_and_content: FPKM gene expression in CSV format.
 
Submission date May 17, 2016
Last update date Mar 12, 2018
Contact name Lao H Saal
E-mail(s) lao.saal@med.lu.se
Organization name Lund University
Department Department of Oncology and Pathology
Lab Translational Oncogenomics Unit
Street address Scheelevägen 2, MV404B2
City Lund
ZIP/Postal code 22391
Country Sweden
 
Platform ID GPL11154
Series (2)
GSE81538 Clinical Value of RNA Sequencing–Based Classifiers for Prediction of the Five Conventional Breast Cancer Biomarkers: A Report From the Population-Based Multicenter Sweden Cancerome Analysis Network—Breast Initiative [cohort 405]
GSE81540 Clinical Value of RNA Sequencing–Based Classifiers for Prediction of the Five Conventional Breast Cancer Biomarkers: A Report From the Population-Based Multicenter Sweden Cancerome Analysis Network—Breast Initiative [superseries]
Relations
BioSample SAMN05006248

Supplementary data files not provided
Raw data not provided for this record
Processed data are available on Series record

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