Effect of RNA integrity on uniquely mapped reads in RNA-Seq

BMC Research Notes

James A Knowles³  Danea F Rebolini¹  Valeria N Spitsyna³  Oleg V Evgrafov³  Jennifer S Herstein³  Tade Souaiaia³  Emily A Chen² 

[1] Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA;Department of Genetic, Molecular, and Cellular Biology, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, 1501 San Pablo Street, ZNI 401, MC 2821, Los Angeles, CA 90089-2821, USA;Department of Psychiatry and the Behavioral Sciences, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, 1501 San Pablo Street, ZNI 401, MC 2821, Los Angeles, CA 90089-2821, USA

关键词: Degradation;    Library construction protocol;    Low-quality RNA;    RNA-Sequencing;    Gene expression;   

Others  :  1125829
DOI  :  10.1186/1756-0500-7-753

 received in 2014-03-20, accepted in 2014-10-14,  发布年份 2014

【 摘 要 】

Background

We examined the performance of three RNA-Sequencing library preparation protocols as a function of RNA integrity, comparing gene expressions between heat-degraded samples to their high-quality counterparts. This work is invaluable given the difficulty of obtaining high-quality RNA from tissues, particularly those from individuals with disease phenotypes.

Results

With the integrity of total RNA being a critical parameter for RNA-Sequencing analysis, degraded RNA can heavily influence the results of gene expression profiles. We discovered that gene expression read results are influenced by RNA quality when a common library construction protocol is used. These results are based on one technical experiment from a pool of 4 neural progenitor cell lines.

Conclusions

The use of alternative protocols can allow samples with a wider range of RNA qualities to be used, facilitating the investigation of disease tissues.

【 授权许可】

   
2014 Chen et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Adiconis X, Borges-Rivera D, Satija R, DeLuca DS, Busby MA, Berlin AM, Sivachenko A, Thompson DA, Wysoker A, Fennell T, Gnirke A, Pochet N, Regev A, Levin JZ: Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat Methods 2013, 10:623-629.
• [2]Evgrafov OV, Wrobel BB, Kang X, Simpson G, Malaspina D, Knowles JA: Olfactory neuroepithelium-derived neural progenitor cells as a model system for investigating the molecular mechanisms of neuropsychiatric disorders. Psychiatr Genet 2011, 21:217-228.
• [3]Opitz L, Salinas-Riester G, Grade M, Jung K, Jo P, Emons G, Ghadimi BM, Beissbarth T, Gaedcke J: Impact of RNA degradation on gene expression profiling. BMC Med Genomics 2010, 3:36. BioMed Central Full Text
• [4]Chen Y, Souaiaia T, Chen T: PerM: efficient mapping of short sequencing reads with periodic full sensitive spaced seeds. Bioinformatics 2009, 25:2514-2521.
• [5]Trapnell C, Pachter L, Salzberg S: TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 2009, 25:1105-1111.
• [6]Anders S, Pyl PT, Huber W: HTSeq - A Python framework to work with high-throughput sequencing data. BioRxiv preprint 2014. http://www.ncbi.nlm.nih.gov/pubmed/25260700 webcite