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  • graceqy
    replied
    Got it. Thanks!

    Leave a comment:

  • fanli
    replied
    I don't see why that'd be a problem...

    Quoting from this benchmark paper:
    A comprehensive evaluation of ensembl, RefSeq, and UCSC annotations in the context of RNA-seq read mapping and gene quantification - BMC Genomics
    http://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-015-1308-8
    Background RNA-Seq has become increasingly popular in transcriptome profiling. One aspect of transcriptome research is to quantify the expression levels of genomic elements, such as genes, their transcripts and exons. Acquiring a transcriptome expression profile requires genomic elements to be defined in the context of the genome. Multiple human genome annotation databases exist, including RefGene (RefSeq Gene), Ensembl, and the UCSC annotation database. The impact of the choice of an annotation on estimating gene expression remains insufficiently investigated. Results In this paper, we systematically characterized the impact of genome annotation choice on read mapping and transcriptome quantification by analyzing a RNA-Seq dataset generated by the Human Body Map 2.0 Project. The impact of a gene model on mapping of non-junction reads is different from junction reads. For the RNA-Seq dataset with a read length of 75 bp, on average, 95% of non-junction reads were mapped to exactly the same genomic location regardless of which gene models was used. By contrast, this percentage dropped to 53% for junction reads. In addition, about 30% of junction reads failed to align without the assistance of a gene model, while 10–15% mapped alternatively. There are 21,958 common genes among RefGene, Ensembl, and UCSC annotations. When we compared the gene quantification results in RefGene and Ensembl annotations, 20% of genes are not expressed, and thus have a zero count in both annotations. Surprisingly, identical gene quantification results were obtained for only 16.3% (about one sixth) of genes. Approximately 28.1% of genes’ expression levels differed by 5% or higher, and of those, the relative expression levels for 9.3% of genes (equivalent to 2038) differed by 50% or greater. The case studies revealed that the gene definition differences in gene models frequently result in inconsistency in gene quantification. Conclusions We demonstrated that the choice of a gene model has a dramatic effect on both gene quantification and differential analysis. Our research will help RNA-Seq data analysts to make an informed choice of gene model in practical RNA-Seq data analysis.


    ...when conducting research that emphasizes reproducible and robust gene expression estimates, a less complex genome annotation, such as RefGene, might be preferred. When conducting more exploratory research, a more complex genome annotation, such as Ensembl, should be chosen.

    Leave a comment:

  • graceqy
    started a topic Kallisto with refseq fasta file

    Kallisto with refseq fasta file

    Has anyone ever tried to use refseq instead of ensembl transcriptome fasta files for RNAseq analysis with Kallisto, pls? Would there be any concern for me to do so?
    Tags: None
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