Comprehensively identifying and characterizing the missing gene sequences in human reference genome with integrated analytic approaches.

The human reference genome is still incomplete and a number of gene sequences are missing from it. The approaches to uncover them, the reasons causing their absence and their functions are less explored. Here, we comprehensively identified and characterized the missing genes of human reference genome with RNA-Seq data from 16 different human tissues. By using a combined approach of genome-guided transcriptome reconstruction coupled with genome-wide comparison, we uncovered 3.78 and 2.37 Mb transcribed regions in the human genome assemblies of Celera and HuRef either missed from their homologous chromosomes of NCBI human reference genome build 37.2 or partially or entirely absent from the reference. We further identified a significant number of novel transcript contigs in each tissue from de novo transcriptome assembly that are unalignable to NCBI build 37.2 but can be aligned to at least one of the genomes from Celera, HuRef, chimpanzee, macaca or mouse. Our analyses indicate that the missing genes could result from genome misassembly, transposition, copy number variation, translocation and other structural variations. Moreover, our results further suggest that a large portion of these missing genes are conserved between human and other mammals, implying their important biological functions. Totally, 1,233 functional protein domains were detected in these missing genes. Collectively, our study not only provides approaches for uncovering the missing genes of a genome, but also proposes the potential reasons causing genes missed from the genome and highlights the importance of uncovering the missing genes of incomplete genomes.

Incorporating the human gene annotations in different databases significantly improved transcriptomic and genetic analyses.

Human gene annotation is crucial for conducting transcriptomic and genetic studies; however, the impacts of human gene annotations in diverse databases on related studies have been less evaluated. To enable full use of various human annotation resources and better understand the human transcriptome, here we systematically compare the human annotations present in RefSeq, Ensembl (GENCODE), and AceView on diverse transcriptomic and genetic analyses. We found that the human gene annotations in the three databases are far from complete. Although Ensembl and AceView annotated more genes than RefSeq, more than 15,800 genes from Ensembl (or AceView) are within the intergenic and intronic regions of AceView (or Ensembl) annotation. The human transcriptome annotations in RefSeq, Ensembl, and AceView had distinct effects on short-read mapping, gene and isoform expression profiling, and differential expression calling. Furthermore, our findings indicate that the integrated annotation of these databases can obtain a more complete gene set and significantly enhance those transcriptomic analyses. We also observed that many more known SNPs were located within genes annotated in Ensembl and AceView than in RefSeq. In particular, 1033 of 3041 trait/disease-associated SNPs involved in about 200 human traits/diseases that were previously reported to be in RefSeq intergenic regions could be relocated within Ensembl and AceView genes. Our findings illustrate that a more complete transcriptome generated by incorporating human gene annotations in diverse databases can strikingly improve the overall results of transcriptomic and genetic studies.