title: Comprehensive evaluation of AmpliSeq transcriptome, a novel targeted whole transcriptome RNA sequencing methodology for global gene expression analysis.
abstract: 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.
authors.affiliation: Department of Pediatrics, Section of Genomic Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. wenli@mcw.edu.
authors.forename: Wenli
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Pediatrics, Section of Genomic Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. amturner@mcw.edu.
authors.forename: Amy
authors.initials: A
authors.lastname: Turner
authors.affiliation: Department of Pediatrics, Section of Genomic Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. paggarwal@mcw.edu.
authors.forename: Praful
authors.initials: P
authors.lastname: Aggarwal
authors.affiliation: Department of Pediatrics, Section of Genomic Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. amatter@mcw.edu.
authors.forename: Andrea
authors.initials: A
authors.lastname: Matter
authors.affiliation: Department of Pediatrics, Section of Genomic Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. estorvick@mcw.edu.
authors.forename: Erin
authors.initials: E
authors.lastname: Storvick
authors.affiliation: Department of Epidemiology, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham, AL, 35294, USA. arnett@uab.edu.
authors.forename: Donna K
authors.initials: DK
authors.lastname: Arnett
authors.affiliation: Department of Pediatrics, Section of Genomic Pediatrics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA. ubroeckel@mcw.edu.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Broeckel
mesh_terms: D016000:Cluster Analysis; D019295:Computational Biology / Q000379:methods; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000379:methods; D059467:Transcriptome