calculate z-score from rpkm values.

Question: calculate z-score from rpkm values.

5.6 years ago by

vyom84 • 30

United States

vyom84 • 30 wrote:

Hello everyone,

The RNA-seq data from TCGA contain the Z-SCORE, instead of RPKM values. I wanted to perform analysis of TCGA data and GTEx data. But the problem is GTEx data contains RPKM and counts values. There is a tutorial explaining the Z-score calculation (http://dept.stat.lsa.umich.edu/~kshedden/Python-Workshop/gene_expression_comparison.html), but i am not sure it works for RNA-seq or it is the correct tutorial.

Can someone please guide me how to calculate the z-score of genes using GTEx RNA-seq data (RPKM and count data). If possible verify the above link, is it correct code.

Thanks

gtex rna-seq tcga z-score • 10k views

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modified 5.6 years ago • written 5.6 years ago by vyom84 • 30

Where are you downloading your TCGA data? TCGA Data Portal or elsewhere? TCGA provides 2 types of RNAseq data. The older pipeline is just called RNAseq, while the newer pipeline is referred to as RNAseqV2. Which one are you using? Both version provide the raw counts as well as RPKM for the old pipeline and RSEM scaled estimate counts for the RNAseqV2 data. These data are not z-scores. Please see this site for more details on RNAseqV2 data: https://wiki.nci.nih.gov/display/TCGA/RNASeq+Version+2

ADD REPLY • link written 5.6 years ago by alolex • 910

Thanks for the reply and the link.

I have download the data directly from COSMIC. Below is the head of the expression file:

ID_SAMPLE   SAMPLE_NAME   GENE_NAME   REGULATION   Z_SCORE   ID_STUDY
1337808   TCGA-02-2483-01   SFMBT1   over   2.416   329
1337808   TCGA-02-2483-01   SGCE   normal   -0.274   329
1337808   TCGA-02-2483-01   RCAN2   normal   -0.577   329
1337808   TCGA-02-2483-01   KIAA0895   normal   -0.333   329
1337808   TCGA-02-2483-01   LIN7C   normal   0.428   329
1337808   TCGA-02-2483-01   NDUFV2   normal   0.790   329
1337808   TCGA-02-2483-01   AKAP14   normal   -0.275   329
1337808   TCGA-02-2483-01   ST20   normal   0.808   329
1337808   TCGA-02-2483-01   CLCF1   normal   -0.640   329

I think z-score is easy to understand and explain. So, i wanted to convert the GTEx data (counts or RPKM) values into z-score. Please advised how to convert the GTEx data into z-score, i have both the reads and counts. Below is the sample of GTEx data:

TargetID   Gene_Symbol   Chr   Coord   GTEX-N7MS-0007-SM-2D7W1   GTEX-N7MS-0008-SM-4E3JI   GTEX-N7MS-0011-R10A-SM-2HMJK   GTEX-N7MS-0011-R11A-SM-2HMJS   GTEX-N7MS-0011-R1a-SM-2HMJG   GTEX-N7MS-0011-R2a-SM-2HML6
ENST00000390859.1   ENSG00000212161.1   1   159821762   0.000000   0.000000   0.000000   0.000000   0.000000   0.000000
ENST00000290551.4   ENSG00000159388.5   1   203274619   4356.000000   2685.999512   6367.449707   9156.000000   4144.000000   5143.111328
ENST00000475157.1   ENSG00000159388.5   1   203274619   0.000000   18.000416   8.550518   0.000000   0.000000   32.888611
ENST00000429660.1   ENSG00000223683.1   1   63727784   0.000000   0.000000   0.000000   0.000000   0.000000   0.000000
ENST00000473120.1   ENSG00000092850.7   1   36549676   0.000000   0.000000   19.124006   18.559824   0.000000   23.603249
ENST00000207457.3   ENSG00000092850.7   1   36549676   0.000000   0.000000   112.875992   53.440178   207.776093   112.396751
ENST00000469024.1   ENSG00000092850.7   1   36549676   0.000000   0.000000   0.000000   0.000000   52.223907   0.000000
ENST00000489568.1   ENSG00000162461.7   1   16062900   0.000000   0.000000   0.000000   0.000000   0.000000   0.000000
ENST00000294454.5   ENSG00000162461.7   1   16062900   68.000000   132.000000   696.000000   5004.000000   512.000000   312.000000

Thanks

ADD REPLY • link written 5.6 years ago by vyom84 • 30

I don't use COMIC much, so I researched this a little. My thought would be to calculate the z-score like COSMIC does, but the problem is that the help file they link to for more information is broken. You may want to contact them about this. Then I found this Biostars answer by @David Fredman TCGA: What are mRNA expression z-scores? Does TCGA have mRNA expression from controls? , which I think answers your question. It looks like COSMIC uses the z-score so that they can have comparable gene expression across various platforms in TCGA; however, it is not clear if COSMIC calculated them or they were taken straight from TCGA. I think this is a good way to go since you are comparing yet another platform. However, note in the response by @David Fredman that TCGA uses the distribution of the normal tissues (sometimes!) as the control distribution. You should be careful comparing these z-scores to your data if you do not have a comparable control to use because you will get false positive results. My suggestions would be to 1) contact COSMIC to see how they calculated theses z-scores and 2) if that doesn't work out to download the original TCGA count data and calculate the z-scores yourself so that you know what is being used as the control distribution.

ADD REPLY • link written 5.6 years ago by alolex • 910

Just out of curiosity, are you trying to compare GTEx & TCGA? If yes, then why not use only TCGA as it already has data for matched Normal-Tumor samples.

ADD REPLY • link modified 5.6 years ago • written 5.6 years ago by komal.rathi • 3.7k

TCGA's "tumor adjacent" normals for rna-seq are not perfectly normal, due to lymphocyte infiltration or some other tumor microenvironment or epigenetic effects that need further investigation.

ADD REPLY • link modified 5.6 years ago • written 5.6 years ago by Cyriac Kandoth ♦ 5.5k

Never gave it a thought. Thanks!

ADD REPLY • link written 5.6 years ago by komal.rathi • 3.7k

5.6 years ago by

Sean Davis ♦ 26k

National Institutes of Health, Bethesda, MD

Sean Davis ♦ 26k wrote:

You can calculate a z-score by subtracting the mean and dividing by the standard deviation for each gene. You may want to transform your data before doing this (log-transform or other).

ADD COMMENT • link written 5.6 years ago by Sean Davis ♦ 26k

It should probably be pointed out that while this will yield z-scores, the scores themselves may or may not be very meaningful. In fact, unless all of the samples have been normalized in a meaningful way ahead of time the resulting z-scores won't be comparable.

ADD REPLY • link written 5.6 years ago by Devon Ryan ♦ 98k

I totally agree. While the calculation can be done, the utility of doing so remains unknown.

ADD REPLY • link written 5.6 years ago by Sean Davis ♦ 26k

Reading the literature and comments, my understanding of the z-score:

1. Convert the count/RPKM values of each gene into log values.

2. Calculate the mean and standard deviation of X gene log values in 20 lung tissues (suppose i have data for 20 samples).

3. For first lung tissue sample: (gene X log value - mean of log values of 20 lung tissues)/ standard deviation of log values of 20 lung tissues.

4. Now. i have the z-score for gene x in first lung tissue sample. Using the above protocol, i can convert all genes log values into z-score.

The question is the above protocol is correct or not, please advised.

Should i calculate the z-score using reads count or RPKM values.

Does these z-score really have meaning. The z-score COSMIC provide:

ID_SAMPLE   SAMPLE_NAME   GENE_NAME   REGULATION   Z_SCORE   ID_STUDY
1337808   TCGA-02-2483-01   SFMBT1   over   2.416   329
1337808   TCGA-02-2483-01   SGCE   normal   -0.274   329

If i calculate the z-score using above approach, should i be able to calculate the z-score and find out whether the gene is over regulated or normal regulated .

Please advised how to proceed.

Thankx

ADD REPLY • link written 5.6 years ago by vyom84 • 30

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