Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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?

  • #2
    I don't see why that'd be a problem...

    Quoting from this benchmark paper:
    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.

    Comment


    • #3
      Got it. Thanks!

      Comment

      Latest Articles

      Collapse

      • seqadmin
        Strategies for Sequencing Challenging Samples
        by seqadmin


        Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
        03-22-2024, 06:39 AM
      • seqadmin
        Techniques and Challenges in Conservation Genomics
        by seqadmin



        The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

        Avian Conservation
        Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
        03-08-2024, 10:41 AM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, 03-27-2024, 06:37 PM
      0 responses
      12 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-27-2024, 06:07 PM
      0 responses
      11 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-22-2024, 10:03 AM
      0 responses
      53 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-21-2024, 07:32 AM
      0 responses
      68 views
      0 likes
      Last Post seqadmin  
      Working...
      X