Comprehensive evaluation of AmpliSeq transcriptome, a novel targeted whole transcriptome RNA sequencing methodology for global gene expression analysis.

Wenli Li,Ulrich Broeckel,Andrea Matter,Erin Storvick,Amy Turner,Donna K Arnett,Praful Aggarwal

doi:10.1186/s12864-015-2270-1

Wenli Li, Ulrich Broeckel + Show 5 more

Open Access

https://doi.org/10.1186/s12864-015-2270-1

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Abstract

BackgroundWhole 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).ResultsUsing 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2270-1) contains supplementary material, which is available to authorized users.

Highlights

Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis
Our analysis consistently suggests that AmpliSeq performs as well as whole transcriptome sequencing (RNA-seq) for gene expression quantification
Because of the targeted nature and small amplicon size, the total number of raw reads needed for differentially expressed genes (DEGs) analysis for each library prepared with AmpliSeq is much smaller than typical whole-transcriptome RNA sequencing

Summary

Introduction

Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. The Ion AmpliSeqTM 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. RTqPCR has been considered the gold-standard for gene expression quantification with high accuracy and sensitivity [11]. This method can be used to measure the abundance of a single transcript by measuring fluorescent signal intensity through a real-time PCR system. Substantial amount of RNA may be required if a large number of genes need to be tested. This imposes some major hurdles for the highthroughput usage of RT-qPCR

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