Hello, I am having an error running DESeq2 (specifically on the DESeqDataSetFromTximport step) after pseudo-aligning my transcriptomic data with Kallisto. I got the code below from this source: UT Austin Wiki

R

#load libraries
library(tximport)
library("DESeq2")


#Import a file called file_list with all the locations of the abundance.tsv files
#eg below:
#/stor/SCRATCH/sample1/abundances.tsv
#/stor/SCRATCH/sample2/abundances.tsv
#/stor/SCRATCH/sample3/abundances.tsv
#/stor/SCRATCH/sample4/abundances.tsv
files<-as.character(read.table("path/to/file_list.txt", header=FALSE)$V1)
#Import a file called samples with the sample names corresponding to each file in the file_list
#eg below:
#sample1
#sample2
#sample3
#sample4
#look at the data structures
files
#OUTPUT: 
#[1] "/path/to/BMRNA2_sulcia_kallisto/abundance.tsv"    
#[2] "/path/to/BMRNA4_sulcia_kallisto/abundance.tsv"    
#I truncated this because I have 50 samples total
#[50] "/path/to/BMRNANo14_sulcia_kallisto/abundance.tsv"

samples<-as.character(read.table("/path/to/samples.txt",header=FALSE)$V1)
names(files)<-samples
#look at the data structures
samples
#OUTPUT: 
#[1] "BMRNA2_sulcia_kallisto"     "BMRNA4_sulcia_kallisto" 
#truncated again...
#[49] "BMRNANo13c_sulcia_kallisto" "BMRNANo14_sulcia_kallisto"
files
#OUTPUT:
#BMRNA2_sulcia_kallisto 
#   "/Users/mckinleesalazar/Desktop/Dis_stuff/Data/sulcia_transcript/sulcia/just_tsv/BMRNA2_sulcia_kallisto/abundance.tsv"

#Import a file called sampletable which is a tab-delimited file that contains each samplename along with the condition
#eg below:
#samples        condition
#sample1        alc
#sample2        alc
#sample3        con
#sample4        con
sampleTable <-read.table("/path/to/sampletable.txt",header=TRUE, row.names=1)
#look at the data structure
head(sampleTable)
#OUTPUT: 
#Condition
#BMRNA2_sulcia_kallisto   Control1
#BMRNA4_sulcia_kallisto   Control2
#BMRNA12_sulcia_kallisto  Control3
#BMRNA13_sulcia_kallisto  Control4
#BMRNA14_sulcia_kallisto  Control5
#BMRNA20_sulcia_kallisto  Control6


#IMPORTANT: MAKE SURE THE SAMPLES AND FILE_LIST ARE IN THE SAME ORDER- SAMPLES SHOULD MATCH UP WITH FILES
samples==rownames(sampleTable) #should return TRUE for all
#OUTPUT: 
#[1] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
#[19] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
#[37] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE


#Import a file called tx2gene.csv which a csv file that contains the transcript id to gene id mapping
#For Drosophila, this is located at: tx2gene.csv
tx2gene <- read.csv("/path/to/tx2gene.csv")

#Sample of what the .csv format 
#TXNAME,GENEID
#SULC_00001,50S ribosomal protein L35
#SULC_00002,Chaperone protein DnaK
#SULC_00003,50S ribosomal protein L13
#SULC_00004,30S ribosomal protein S9
#SULC_00005,30S ribosomal protein S2
#SULC_00006,tRNA-Leu(taa)
#SULC_00007,tRNA-Gly(gcc)

#look at this data structure
#read in kallisto abundance files, summarizing by gene
txi <- tximport(files, type = "kallisto", tx2gene = tx2gene)
names(txi)
#OUTPUT: 
#[1] "abundance"           "counts"              "length"              "countsFromAbundance"


#make a deseq2 object from the kallisto summarized counts
ddsMatrix <- DESeqDataSetFromTximport(countData = txi, colData = sampleTable, design = ~Condition)
ddsMatrix
#OUTPUT ERROR MESSAGE: 
#Error in is(txi, "list") : argument "txi" is missing, with no default

I am not very experienced in R, but is the issue with the txi data table? I have included a screenshot of what it looks like when I view it in R studios. Is this the correct format? I.e., Should all the counts data be in a single column/row like it appears in the viewer? This doesn't seem right to be given the line:

DESeqDataSetFromTximport(countData = txi, colData = sampleTable, design = ~Condition)

It looks like it is finding the column data from 'sampleTable' but is not able to find the count data from txi. This is why I think it is a format issues in 'txi', but I cannot find an example of the expected format for the txi table.

I would appreciate any help/guidance from an experienced R user. Thanks, McKinlee

argument txi is missing, with no default

This means the function was expecting a named argument txi

Check the man page for the function:

?functionName

You can see a usage example that way

The arguments for that function can be found in the help page (accessed using ?DESeqDataSetFromTximport)

Usage:

     DESeqDataSet(se, design, ignoreRank = FALSE)

     DESeqDataSetFromMatrix(
       countData,
       colData,
       design,
       tidy = FALSE,
       ignoreRank = FALSE,
       ...
     )

     DESeqDataSetFromHTSeqCount(
       sampleTable,
       directory = ".",
       design,
       ignoreRank = FALSE,
       ...
     )

     DESeqDataSetFromTximport(txi, colData, design, ...)

And you can see that there is no argument called 'countData', but there is one called 'txi'. You need to change your function call to use 'txi = txi' instead of 'countData = txi'.