Question: Kallisto/Sleuth DTE analysis - LRT test results in more DTE compared to Wald test.
0
gravatar for MK
11 months ago by
MK0
Czech Republic/Olomouc/IEB
MK0 wrote:

Hello everyone, I am trying to get differential transcript/gene expression using sleuth on my dataset which consist of 3 replicates of well watered and not watered plants. I am using kallisto/sleuth pipeline. I realized I am getting more significant hits (qval <= 0.05) after running LRT test compared to Wald test. Isn't it supposed to be opposite? I read that LRT is a more stringent test. There is a difference of over 5000 transcripts between them!

so <- sleuth_prep(s2c, target_mapping = t2g, ~ condition, extra_bootstrap_summary = TRUE)
so <- sleuth_fit(so, ~condition, 'full')
so <- sleuth_fit(so, ~1, 'reduced')
so <- sleuth_lrt(so, 'reduced', 'full')
so <- sleuth_wt(so,'conditionwater','full')
results_table_lrt <- sleuth_results(so, 'reduced:full', 'lrt', show_all = FALSE)
results_lrt_significant <- results_table_lrt$target_id[which(results_table_lrt$qval <= 0.05)]
results_table_wald <- sleuth_results(so, 'conditionwater','full', test_type = 'wt')
results_wald_significant <- results_table_wald$target_id[which(results_table_wald$qval <= 0.05)]

table(results_table_lrt$qval<=0.05)
FALSE TRUE
53234 32237
table(results_table_wald$qval<=0.05)
FALSE TRUE
57549 27922

I would greatly appreciate any help.

rna-seq sleuth • 510 views
ADD COMMENTlink written 11 months ago by MK0

How many levels does condition have?

ADD REPLYlink written 11 months ago by Devon Ryan94k

This is my metadata file.

sample  condition 
D4_1    drought
D4_2    drought
D4_3    drought
W4_1    water
W4_2    water
W4_3    water
ADD REPLYlink modified 11 months ago • written 11 months ago by MK0

I vaguely remember the original sleuth publication recommending LRT over Wald for some reason. If my recollection is correct then whatever the reason behind that was is likely the cause. I think sleuth has an email list, so you might ask on there.

ADD REPLYlink written 11 months ago by Devon Ryan94k

I think you are right. The reason why I want to run it together with Wald test is to be able to get beta value. I don't know how to get any estimation of fold change while running only LRT test on its own.

ADD REPLYlink written 11 months ago by MK0

[comment not directly related to the question] : ~30K transcript with qval <= 0.05 seems quiet a lot. Is this expected for watered/non-watered plants ?

ADD REPLYlink written 11 months ago by Nicolas Rosewick8.7k

I might be wrong, but as wheat has a huge genome and drought stress will probably affect a lot of pathways this should be fine. Nevertheless, I am not experienced in working with wheat and this is my first RNA-seq analysis.

ADD REPLYlink written 11 months ago by MK0
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