Evaluation of the coverage and depth of transcriptome by RNA-Seq in chickens

BMC Bioinformatics

Proceedings

Charles D Johnson¹  Noushin Ghaffari²  Ulisses M Braga-Neto³  Rui Chen⁴  Hui Wang⁴  Huaijun Zhou⁵  Ying Wang⁵ 

[1] AgriLife Genomics and Bioinformatics Services, Texas A&M University, 77843-2312, College Station, TX, USA;AgriLife Genomics and Bioinformatics Services, Texas A&M University, 77843-2312, College Station, TX, USA;Department of Electrical and Computer Engineering, Texas A&M University, 77843, College Station, TX, USA;Department of Electrical and Computer Engineering, Texas A&M University, 77843, College Station, TX, USA;Department of Molecular and Human Genetics, Baylor College of Medicine, 77030, Houston, TX, USA;Department of Poultry Science, Texas A &M University College Station, 77843-2472, TX, USA;

关键词: Sequencing Depth;    Chicken Genome;    Illumina Genome Analyzer;    Quartile Group;    Transcriptome Coverage;   

DOI  :  10.1186/1471-2105-12-S10-S5

来源: Springer

【 摘 要 】

BackgroundRNA-Seq is the recently developed high-throughput sequencing technology for profiling the entire transcriptome in any organism. It has several major advantages over current hybridization-based approach such as microarrays. However, the cost per sample by RNA-Seq is still prohibitive for most laboratories. With continued improvement in sequence output, it would be cost-effective if multiple samples are multiplexed and sequenced in a single lane with sufficient transcriptome coverage. The objective of this analysis is to evaluate what sequencing depth might be sufficient to interrogate gene expression profiling in the chicken by RNA-Seq.ResultsTwo cDNA libraries from chicken lungs were sequenced initially, and 4.9 million (M) and 1.6 M (60 bp) reads were generated, respectively. With significant improvements in sequencing technology, two technical replicate cDNA libraries were re-sequenced. Totals of 29.6 M and 28.7 M (75 bp) reads were obtained with the two samples. More than 90% of annotated genes were detected in the data sets with 28.7-29.6 M reads, while only 68% of genes were detected in the data set with 1.6 M reads. The correlation coefficients of gene expression between technical replicates within the same sample were 0.9458 and 0.8442. To evaluate the appropriate depth needed for mRNA profiling, a random sampling method was used to generate different number of reads from each sample. There was a significant increase in correlation coefficients from a sequencing depth of 1.6 M to 10 M for all genes except highly abundant genes. No significant improvement was observed from the depth of 10 M to 20 M (75 bp) reads.ConclusionThe analysis from the current study demonstrated that 30 M (75 bp) reads is sufficient to detect all annotated genes in chicken lungs. Ten million (75 bp) reads could detect about 80% of annotated chicken genes, and RNA-Seq at this depth can serve as a replacement of microarray technology. Furthermore, the depth of sequencing had a significant impact on measuring gene expression of low abundant genes. Finally, the combination of experimental and simulation approaches is a powerful approach to address the relationship between the depth of sequencing and transcriptome coverage.

【 授权许可】

Unknown   
© Wang et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.