# error in random forest analysis

I am now struggling to do random forest analysis, I will be thankful if you could help with code for random forest analysis.

I got samples from the root, soil, and leaf from two regions (bau & mau) and these samples belong to two seasons (Wet and Dry).

Now I am interested to do random forest analysis at genera or family level to identify the taxa which contribute the differences like in root samples based on region as well as season.

Here is my code, but I am getting the error.

    library(randomForest)
library(knitr)

#### RANDOM FOREST ANALYSIS #####
#### Prepare data ####
table(apply(OTU_table,1,sum)) #verify rarefaction

Meta$sampleid=rownames(Meta) #Load taxonomy taxo=read.table("taxonomy.txt", row.names=1, sep="\t", header=F ,stringsAsFactors=F,quote="") rownames(taxo)=paste("a.",row.names(taxo),sep="") #### Run models #### #1. Root only # 1.1. both region # 1.2. bau only # 1.3. mau only #2. Soil only # 2.1. both region # 2.2. bau only # 2.3. mau only #Params RF NTREE=1000 # Number of Trees NbVar=1000 # Number of variables tested at each split #### Root ONLY 1-3 #### # 1. BOTH Region #Subset of data RootSamples=as.character(Meta[Meta$Compartment=="Root","sampleid"])
Root_OTU_table=OTU_table[RootSamples,]

#Model with microbiome based on Season, region

whole_root_pred=data.frame(Season=Meta[RootSamples,"Season"],Region=Meta[RootSamples,"Region"],a=Root_OTU_table)

Season Region a.d2ec9f3b77975c0f457e4b7413b217ff

**RF_model_Root_all=randomForest(y=?,sampsize=c(143,143),strata=?,x=whole_Root_pred,importance = T,proximity = T,ntree =
NTREE,mtry = NbVar)**

print(RF_model_Root_all)
#plot summary using the 5% most important OTUs ERROR ON LAST LINE
imp=data.frame(importance(RF_model_Root_all))
imp$$genus=as.character(taxo[rownames(imp),"Genus"]) Best=imp[imp$$MeanDecreaseAccuracy>quantile(x = imp\$MeanDecreaseAccuracy,.95),]
bymedian <- with(Best, reorder(genus, -MeanDecreaseAccuracy, median))

pdf(width = 20,height = 10,file=paste(pathforplots,"Variable_Importance_Root_BothRegion_raref.pdf",sep=""))
par(mar=c(15,5,1,1))
boxplot(Best$$MeanDecreaseAccuracy ~ bymedian, data = Best, xlab = "", ylab = "Variable Importance", main = paste("Root in Both Countries; Error Rate=",round(RF_model_Feces_all$$err.rate[NTREE,"OOB"],3),sep=""), varwidth = TRUE,
col = "lightgray",las=2)

dev.off()


Many thanks

For example, we will need to see your dataset (whole_root_pred), to decide why Stunting_Root is NULL.
1. You might need to initialize Stunting_Root as a variable. It is currently not clear if it is e.g. a column of your dataframe, or just uninitialized. Uninitialized variables are NULL, which would explain your problem. randomForest might not know to look for strata inside your dataframe, for example. Is it in your dataframe?
2. Also, I might be missing something, but why are you passing ? as a response? I'm not an expert but I believe that is an illegal character in R (I'm pretty sure?).
• @bioinfonext I would recommend looking at the randomForest docs (?randomForest) to decide what to put in these arguments. y is supposed to be the response variable, so either a vector of categories (if task is classification) or a vector of numerics (if task is regression). Again, it would be very helpful to see a sample of the data, both the response variable and the predictor variables, and how they are represented in the code you provide. Oct 12 '20 at 20:42
• head() should be fine. Oct 13 '20 at 18:57