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| Status |
Public on Feb 11, 2016 |
| Title |
Comprehensive Evaluation of AmpliSeq Transcriptome, a Novel Targeted Whole Transcriptome RNA Sequencing Methodology for Global Gene Expression Analysis. |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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 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.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.
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| Overall design |
Comprehensive, performance evaluation of AmpliSeq Transcriptome to standard whole-transcriptome RNA-sequencing methods for large-scale, genome-wide differential gene expression analysis. We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs).
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| Contributor(s) |
Li W, Turner A, Aggarwal P, Matter A, Storvick E, Arnet D, Broeckel U |
| Citation(s) |
26673413 |
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| Submission date |
Nov 06, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Wenli Li |
| E-mail(s) |
wenli@mcw.edu
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| Phone |
4149552381
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| Organization name |
Medical College of Wisconsin
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| Department |
Pediatrics
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| Street address |
8701 Watertown Plank Road
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| City |
Milwaukee |
| State/province |
Wisconsin |
| ZIP/Postal code |
53226 |
| Country |
USA |
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| Platforms (1) |
| GPL17303 |
Ion Torrent Proton (Homo sapiens) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA301430 |
| SRA |
SRP065892 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE74760_RAW.tar |
1.0 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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