FFPE-Fresh RNA-seq HG Normalization

Question

[deniz]

Hello,

I am trying to normalize housekeeping genes for FFPE tumor samples and fresh frozen control samples.

I am both using the Galaxy platform and Linux command line.

Any related comments will be much appreciated

Bests,

Deniz

[ATpoint]

Use any of the established normalization methods, such as TMM in edgeR or the geometric mean approach in DESeq2. The manuals will tell you how to use them.

[deniz]

Thank you for your reply. I have actually tried both DESeq2 and edgeR tools and used HISAT2 for alignment.

However, the housekeeping gene fold change difference did not correlate good enough between FFPE and fresh frozen samples.

As far as I know, people are using geNorm and NormFinder algorithms to estimate the stability of the reference genes.

Is there any way to improve the correlation of genes between these two types of samples?

[ATpoint]

Can you give some details on how you compared your samples and what did not correlate good enough means?

[deniz]

I started the analysis at the Galaxy platform. Since I had limited storage I used 3 FFPE tumors and 3 Fresh Frozen control paired-end samples first and then performed

Below table demonstrates the DESeq2 results of FFPE samples as first-factor level and fresh samples as second factor level.

>  GeneID   Base mean   log2(FC)    StdErr  Wald-Stats  P-value P-adj
> HK_Gene1  2,651,251,265   -1,754,673,442  0,337058353 -5,205,844,711  1.93E-02    1.67E-01
> HK_Gene2  8,186,395,434   -0,34488023 0,535679859 -0,643817804    0,519693587 0,635193033
> HK_Gene3  8,849,195,235   -1,901,222,022  0,259466438 -7,327,429,451  2.35E-08    5.27E-07
> HK_Gene4  8,566,608,327   -0,681018883    0,28779231  -2,366,355,391  0,017964191 0,043898112
> HK_Gene5  2,414,044,572   -1,362,857,524  0,242603863 -561,762,499    1.94E-05    2.02E-02
> HK_Gene6  1,830,798,049   -1,222,550,059  0,272857925 -4,480,537,114  7.45E-01    4.68E+00
> HK_Gene7  1,221,328,248   -2,067,919,636  0,224752083 -9,200,891,962  3.55E-15    1.70E-13

[ATpoint]

What are those samples? Are they supposed to be comparable? What separates the FFPE and fresh samples beyond the preservation method? You should check for batch-effects related to the status being FFPE/fresh. Probably (most likely) there is one.