Am I getting TPM correct from tcga?

Question

[Znow]

Hello everyone,

I am using the TCGAbiolinks package in R to retrieve TPM data from the TCGA database. Below is the code I am using:

library("TCGAbiolinks")

query_TCGA <- GDCquery(
  project = "TCGA-HNSC",
  data.category = "Transcriptome Profiling", 
  data.type = "Gene Expression Quantification",
  experimental.strategy = "RNA-Seq",
  workflow.type = "STAR - Counts"
)

GDCdownload(query = query_TCGA)

tcga_data <- GDCprepare(query_TCGA)

TPM <- assay(tcga_data, 4)

Could you please confirm if I am correctly extracting TPM values with this approach? Specifically, I'm using the assay() function to extract data from tcga_data, assuming that TPM values are in the fourth assay slot, as its name is tpm_unstrand. Is this the correct method to obtain TPM values from TCGA data using TCGAbiolinks?

Thank you for your assistance!

Answer 1

[bk11]

You can get TPM as follows:

library("TCGAbiolinks")

query_TCGA <- GDCquery(
  project = "TCGA-HNSC",
  data.category = "Transcriptome Profiling", 
  data.type = "Gene Expression Quantification",
  experimental.strategy = "RNA-Seq",
  workflow.type = "STAR - Counts"
)

GDCdownload(query = query_TCGA)

tcga_data <- GDCprepare(query_TCGA)

tcga_data
class: RangedSummarizedExperiment 
dim: 60660 566 
metadata(1): data_release
assays(6): unstranded stranded_first ... fpkm_unstrand fpkm_uq_unstrand
rownames(60660): ENSG00000000003.15 ENSG00000000005.6 ... ENSG00000288674.1 ENSG00000288675.1
rowData names(10): source type ... hgnc_id havana_gene
colnames(566): TCGA-CR-7370-01A-11R-2132-07 TCGA-CR-6484-01A-11R-1873-07 ... TCGA-H7-8501-01A-11R-2403-07
  TCGA-CV-7415-01A-11R-2081-07
colData names(80): barcode patient ... paper_Copy.Number paper_PARADIGM

tpm <- tcga_data@assays@data$tpm_unstrand
rownames(tpm) <- rownames(tcga_data)
colnames(tpm) <- colnames(tcga_data)
tpm[1:5, 1:5]
                   TCGA-CR-7370-01A-11R-2132-07 TCGA-CR-6484-01A-11R-1873-07 TCGA-CR-6478-01A-11R-1873-07 TCGA-CV-A45Y-01A-11R-A24Z-07 TCGA-UF-A719-01A-12R-A34R-07
ENSG00000000003.15                      59.7559                      12.9848                      13.4885                      19.7511                      56.4732
ENSG00000000005.6                        0.0000                       0.0000                       0.0295                       0.0000                       0.0000
ENSG00000000419.13                      70.1836                      91.8942                      76.2891                      59.1658                      71.4131
ENSG00000000457.14                       9.9460                       4.5738                       4.8244                       3.7459                       8.0012
ENSG00000000460.17                      14.1288                       5.4911                       8.8251                       2.4338                       9.8382

[sj]

assay(tcga_data, "tpm_unstrand")

will also work

[Ram]

Yes, if used in the context of bk11's answer, not as a standalone function call. Point is, this should have been a comment, not an answer. I've moved it to a comment this time, please be more mindful in the future.