Isoform resource - Methods summary

The Isoform resource presents genome-wide expression profiles for isoforms based on in-house and GTEx RNA-seq data across a large number of normal tissues. Gene level overviews showing the contribution of isoforms to expression and location across tissues are presented alongside the expression profiles of individual isoforms. IHC protein data is shown for isoforms targeted by isoform specific antibodies, and for the GTEx data the distribution of expression across individual samples based on sex and age is displayed for each isoform. The transcriptomics analysis includes classification on gene and isoform level of tissue specificity and expression based on the isoform expression profiles.

Key publication: “Tissue-based map of the human proteome.” Science 347(6220):1260419.

8408289866

What can you learn from the Isoform resource?

Learn about:

• the expression profiles of individual isoforms
• the tissue specificity of individual isoforms
• the predicted location across tissues based on isoform expression
• sex and age differences in isoform expression
• isoform specific protein expression
• the catalogue of genes with a dominant isoform or other categories based on aggregated data

How has the data been generated?

HPA RNA-seq data

The HPA dataset was generated in-house based on mRNA samples from 40 normal tissues extracted from frozen tissue sections. For a total number of 186 normal tissue samples mRNA sequencing was performed on Illumina HiSeq2000 and 2500 machines (Illumina, San Diego, CA, USA) using the standard Illumina RNA-seq protocol with a read length of 2x100 bases.

GTEx RNA-seq data

The GTEx consortium collects and analyzes multiple human post mortem tissues. RNA-seq data from 36 tissue types was imported from the Genotype-Tissue Expression consortium and mapped based on GTEx_Analysis_v10_RSEMv1.3.3_transcripts_tpm.txt.gz and the resulting TPM values have been included in the Human Protein Atlas for all corresponding genes that could be mapped from Gencode v39 to Ensembl version 109.

The GTEx retina data are based on EyeGEx data from Ratnapriya et al., Nature Genetics 2019 and transcript abundance estimation was performed using Kallisto v0.48.0 using Ensembl version 109 as reference genome.

How has the data been analyzed?

Normalization of isoform-level RNA expression data

For both the HPA and GTEx transcriptomics datasets, transcript TPM values in each sample were first scaled to a sum of 1 million TPM (denoted pTPM) to compensate for the transcripts not included in the analysis. GTEx transcript expression values obtained by quantifying transcript TPM from the GTEx release against GENCODE were kept, while transcripts not present in this annotation will have no GTEx data. Next, the pTPM values of all samples within each data source were normalized separately using Trimmed mean of M values (TMM) to allow for between-sample comparisons. The resulting normalized transcript expression values, denoted nTPM, were calculated for each transcript in every sample. The transcript expression level in each tissue was estimated as the average nTPM of all individual samples from that tissue; for tissues with several subtypes, the maximum of the subtype averages was used. nTPM values below 0.1 are not visualized on the Atlas sections.

What is presented in the resource?

Isoform-level RNA expression data across normal human tissues is presented for all protein-coding genes. Bar plots displaying isoform expression aggregated to gene-level as well as individual isoform expression profiles are shown. The aggregated overview enables toggling between expression of individual isoforms and the predicted location based on the isoforms expressed. Protein data is shown if IHC data with reliability Enhanced or Supported exists for an antibody defined as specific for an isoform of a multi-isoform gene. To fulfill the criteria for specific the antigen used for generation of the antibody must map the sequence of one particular isoform with >=80% identity and all other isoforms with less than 60% identity. Data for individual isoforms is obtained by clicking on an isoform name to the left of the plot which will display the expression profile and the specificity classification for that particular isoform. For isoforms in the GTEx data a boxplot showing the distribution of expression of all included samples, with the possibility to toggle between data grouped by sex or age, is displayed. By clicking on the box plot a larger version appears.

Classification of isoform specificity and expression profiles into gene-level categories was also performed and the categories are displayed to the right of the bar plots.

Due to the mapping between Ensembl and GTEx and discrepancies between the versions used genes without GTEx data and genes with isoforms missing in the GTEx exist.

Overview for the gene PPP1R12C showing the expression of the four individual isoforms as well as the gene-level expression and specificity categories in the top left part.


Expression profile of the PPP1R12C-207 isoform including the specificity category in the top left and the distribution of expression for the GTEx samples by sex for this isoform.

How has the classification of all protein-coding genes been done?

A genome-wide classification of the isoforms in each dataset with regard to tissue specificity has been performed using the between-sample normalized data. The isoforms were classified according to specificity into (i) tissue enriched isoforms with at least fourfold higher expression levels in one tissue type as compared with any other analysed tissue; (ii) group enriched isoforms with enriched expression in a small number of tissues (2 to 5); and (iii) tissue enhanced isoforms where nTPM in one or several tissues is at least fourfold higher than the mean nTPM across all tissues, provided the lowest-expressing enhanced tissue is at least twofold higher than the average of the next three tissues (a gap ratio of ≥ 2)., (iv) low tissue specificity isoforms with nTPM ≥ 1 in at least one tissue but not elevated in any tissue, and (v) not detected isoforms with expression < 1 nTPM in all tissues.

The gene-level tissue specificity shown next to the barplots corresponds to the combined unique specificity categories of all individual isoforms for that gene. Gene-level category levels can include any combination of the isoform specificity categories, but with gene-level enriched corresponding to both tissue enriched and group enriched isoforms.

A gene-level classification of expression based on isoform expression profiles was also performed. This expression category include (i) highly dominant isoform, genes with all, many or some isoforms expressed that have a highly dominant isoform defined as the expression of one isoform corresponding to >=70% of all expression across all tissues, (ii) dominant isoform, genes with all, many or some isoforms expressed, for which one isoform has the highest expression across all tissues but the expression corresponds to <70% in at least 1 tissue , (iii) genes with all, many or some isoforms expressed, but no isoform has the highest expression across all tissues, (iv) single isoform expressed, multi-isoform genes with expression of a single isoform, (v) single isoform gene, genes with only 1 isoform, (vi) no isoform over expression cut-off, genes where none of the isoforms have >=1 nTPM in any tissue.

Genes without data or with missing isoforms in the GTEx data are not part of the classifications.

Data overview