Comprehensive evaluation of AmpliSeq transcriptome, a novel targeted whole transcriptome RNA sequencing methodology for global gene expression analysis. BMC Genomics 2015 Dec 16;16:1069
Date
12/18/2015Pubmed ID
26673413Pubmed Central ID
PMC4681149DOI
10.1186/s12864-015-2270-1Scopus ID
2-s2.0-84949678611 (requires institutional sign-in at Scopus site) 80 CitationsAbstract
BACKGROUND: Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq. To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs).
RESULTS: Using published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson's r = 0.92) and Ion Torrent Proton (Pearson's r = 0.92). We used ROC, Matthew's correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations.
CONCLUSIONS: Our study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy.
Author List
Li W, Turner A, Aggarwal P, Matter A, Storvick E, Arnett DK, Broeckel UAuthors
Ulrich Broeckel MD Chief, Center Associate Director, Professor in the Pediatrics department at Medical College of WisconsinAmy Turner Research Scientist I in the Pediatrics department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
Cluster AnalysisComputational Biology
Gene Expression Profiling
High-Throughput Nucleotide Sequencing
Reproducibility of Results
Sequence Analysis, RNA
Transcriptome
