Wednesday, Mar 25, 2015

".... The decision of whether to use DNA microarrays or RNA-seq seems straightforward and unambiguous. In reality, the two technologies couldn't be more complementary."

The ideas of transcriptome arrays for human, mouse and rat

".... The decision of whether to use DNA microarrays or RNA-seq seems straightforward and unambiguous. In reality, the two technologies couldn't be more complementary."
(Scott Peteron, JCVI Weblog, J. Crag Venter Institute)

The adoption of next generation sequencing (NGS) of cDNA (RNA-seq) for transcript profiling were well documented throughout these years. However, microarrays still play an indispensable role to complement the RNA-seq findings. There are challenges that need to overcome for RNA-seq; including sample input requirements, lengthy protocols for library preparation and the complex, high volume data generated after an experiment.
On the other hand, Affymetrix microarray had a defined and stringent protocol from sample preparation to data generation. Typical run time is 2.5 days for the new Human Transcriptome Array 2.0 (HTA). Coupled with the freely available Transcriptome Analysis Console software (TAC software), biologically relevant results would be ready in an additional day of work.

Alternative splicing is an important event in gene regulation. RNA-seq routinely missed important expression changes4. The limitation of RNA-seq is exacerbated when one is looking for low abundance genes. The significance of these low expressers need deep sequencing coverage to get to an acceptable degree of variance (CV) to warrant a meaningful conclusion2.
n a recent MAQC/ SEQC project meeting (US FDA sequencing quality control consortium); using a classic tissue mixture model, the HTA array delivers the equivalent accuracy in gene expression measurements throughout the transcriptome as sequencing a sample across 2 full lanes on a HiSeq 2000 (i.e. 720 million raw reads). Hence, investigators would be missing important expression changes if the RNA-seq data coverage is not deep enough (e.g. due to cost consideration)5.

Though RNA-seq platform in novel gene discovery and gene quantification provides an alternative technology of choice, microarrays still stand out to be a cost-effective and reproducible way to analyse large number of clinical samples on alternative splicing. RNA-seq in sufficient depth would help to discover transcriptome elements related to a disease process, follow-up studies on large patient sample sets could then utilized the HTA arrays as a reliable screening tool1, 3, 4.

For more information on the Affymetrix HTA "complete solution", please visit the product web page http://www.affymetrix.com/estore/promotions/hta/index.affx



References:
1. Transcriptomic Analysis of PNN- and ESRP1-Regulated Alternative Pre-mRNA Splicing in Human Corneal Epithelial Cells
Jeong-Hoon Joo et al
Invest. Ophthalmol. Vis. Sci. 54:697-707 (2013)


2. Impact of the next-generation sequencing data depth on various biological result inferences
Hou Rui et al
Science China 56:104-109 (2013)


3. An integrative functional genomics approach for discovering biomarkers in schizophrenia
Marquis P. Vawter et al
Briefings in Functional Genomics 10:387-399 (2011)


4. Human transcriptome array for high-throughput clinical studies
WeiHong Xu et al
Proc. Natl. Acad. Sci. 108: 3707-3712 (2011)


5. Affymetrix brochure
P/N EMI02502 Rev3
http://media.affymetrix.com/promotions/hta/pdf/hta_volume1_flyer.pdf

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