Total RNA contribution for Metatranscriptomics Analysis with MaAsLin3

Downstream analysis and statistics MaAsLin
1

Hello,

I am learning to do Metatranscriptomics analysis without paired MGX. I read the paper on comparing DE methods (Statistical approaches for differential expression analysis in metatranscriptomics - PMC) and found it super helpful in terms of the steps necessary for a robust statistical comparison - seems like M3 is the way to go for my analysis. I ran my samples through HUMANn4 to get my gene family outputs (KO, GO, and Level4ECs). However, I’m not entirely sure how to get each

…species’ total RNA contributions as a model covariate.

from the HUMANn output. Would it just be # features from species A / # all features?

I read through the MTX model 3 tutorial on github but it uses DNA copy number / abundances to adjust the data - no mention on MTX data alone without MGX.. other than running MaAsLin3 on raw RNA abundances (which I don’t think is the same as M3 from the paper?).

Thanks!

2

Hi,

Model 3 means that you need both the metatranscriptomics scaled within each species (which you have) as well as a way of estimating the taxonomic abundance from the RNA (via a uniformly expressed housekeeper gene or something). I’m not sure if you have the second based on what you’ve described. In MaAsLin 3, you would then use the feature_specific_covariate field with the taxa table from maaslin3::preprocess_taxa_mtx - let me know if the examples in ?preprocess_taxa_mtx aren’t informative enough.

Will

3

Thanks for your reply Will. My pipeline was first QC (using Kneaddata) then metaphlan4 then HUMANn4 (which I used the output of metaphlan for). As such, my taxonomic abundance would’ve been estimated by metaphlan? If I don’t have a uniformly expressed housekeeper gene spiked in, is there any way to use model 3 still?

4

Sorry, is that to say you put the RNA sequencing through MetaPhlAn?

5

Correct. I ran metaphlan4 separately because if I remember correctly, HUMANn4 invokes an outdated version of metaphlan/chocophlan. So instead, I ran metaphlan4 separately and then passed those output files into humann4 using the --taxonomic_profiles flag.