Transcriptomics

Dataset Information

2

A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequence Quality Control consortium


ABSTRACT: We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the United States Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for sequence discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed, for these and qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcriptlevel profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings. The well-characterized reference RNA samples A (pooled cell lines) and B (human brain) from the MAQC consortium, adding spike-ins of synthetic RNA from the External RNA Control Consortium (ERCC). Samples C and D were then constructed by combining A and B in known mixing ratios, 3:1 and 1:3, respectively. All samples were distributed to several independent sites for RNA-Seq library construction and profiling by Illumina HiSeq 2000 and LifeTech SOLiD 5500 platforms. Also, vendors created their own cDNA libraries that were then distributed to each test site, in order to examine the degree of a “site effect” that was independent of the library preparation process. To support an assessment of gene models, samples A and B were also sequenced at independent sites by the Roche 454 platform, providing longer reads. For comparison to other technologies, these data were also compared to the MAQC-I Affymetrix U133 Plus2 microarray, several current microarray platforms, and also assessed by 20,801 PrimePCR reactions.

ORGANISM(S): Homo sapiens  

SUBMITTER: Xin-Xing Tan   Ching-Wei Chang  Zhiyu Peng  Nadereh Jafari  Roy Chaudhuri  Hans Binder  Wendell D Jones  Tzu-Ming Chu  Joshua Xu  Tao Chen  Weihong Xu  Desmond I Bannon  Pierre R Bushel  Fei Lu  Jo Vandesompele  Edward J Oakeley  Kelli Bramlett  Thomas Blomquist  Wei Shi  Marco Chierici  Zhenqiang Su  Scott S Auerbach  Shashi Amur  Wenzhong Xiao  Djork-Arné Clevert  Geng Chen  Hanlin Gao  Javier Santoyo-Lopez  Robert A Setterquist  Jean Thierry-Mieg  Wenjun Bao  Yang Liao  Weimin Cai  Pawel P Labaj  Gary P Schroth  Cecilie Boysen  Roderick V Jensen  Reagan Kelly  Leming Shi  Charles Wang  Craig A Praul  Andreas Scherer  Huixiao Hong  Sheng Li  Ana Conesa  Jennifer G Catalano  E A Thompson  Danielle Thierry-Mieg  Jiri Zavadil  Murray H Brilliant 

PROVIDER: E-GEOD-56457 | ArrayExpress | 2014-08-08

SECONDARY ACCESSION(S): GSE56457PRJNA243413

REPOSITORIES: GEO, ArrayExpress

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