Combining RNA-Seq read counts from 2 lanes of the same sample (.txt file)

Question

[mohammedtoufiq91]

Hi,

I have a question on combining read counts from 2 lanes of the same sample. I have a very large RNA-Seq dataset downloaded from the NCBI GEO. The data files are in the *.txt format and each sample with 2 lanes obtained from featureCounts. I would like to perform the differential expression analysis using this data. Since, each sample is of 2 lanes, please let me know if these samples can be combined by sum or mean or if there is any other method?

dput(Sample_1_L1_S65_L001_R1_001Aligned.sortedByCoord.out_gene.featureCounts.txt)

structure(list(Geneid = c("ENSG00000223972", "ENSG00000227232", 
                          "ENSG00000278267", "ENSG00000243485", "ENSG00000237613", "ENSG00000268020", 
                          "ENSG00000240361", "ENSG00000186092"), Sample_1_L1_S65_L001_R1_001Aligned.sortedByCoord.out.bam.txt = c(0L, 
                                                                                                                              22L, 1L, 0L, 0L, 4L, 0L, 6L)), class = "data.frame", row.names = c(NA, 
                                                                                                                                                                                                 -8L))
#>            Geneid Sample_1_L1_S65_L001_R1_001Aligned.sortedByCoord.out.bam.txt
#> 1 ENSG00000223972                                                            0
#> 2 ENSG00000227232                                                           22
#> 3 ENSG00000278267                                                            1
#> 4 ENSG00000243485                                                            0
#> 5 ENSG00000237613                                                            0
#> 6 ENSG00000268020                                                            4
#> 7 ENSG00000240361                                                            0
#> 8 ENSG00000186092                                                            6

dput(Sample_1_L1_S65_L002_R1_001Aligned.sortedByCoord.out_gene.featureCounts.txt)

structure(list(Geneid = c("ENSG00000223972", "ENSG00000227232", 
                          "ENSG00000278267", "ENSG00000243485", "ENSG00000237613", "ENSG00000268020", 
                          "ENSG00000240361", "ENSG00000186092"), Sample_1_L1_S65_L002_R1_001Aligned.sortedByCoord.out.bam.txt = c(1L, 
                                                                                                                              27L, 2L, 0L, 0L, 2L, 0L, 1L)), class = "data.frame", row.names = c(NA, 
                                                                                                                                                                                                 -8L))
#>            Geneid Sample_1_L1_S65_L002_R1_001Aligned.sortedByCoord.out.bam.txt
#> 1 ENSG00000223972                                                            1
#> 2 ENSG00000227232                                                           27
#> 3 ENSG00000278267                                                            2
#> 4 ENSG00000243485                                                            0
#> 5 ENSG00000237613                                                            0
#> 6 ENSG00000268020                                                            2
#> 7 ENSG00000240361                                                            0
#> 8 ENSG00000186092                                                            1

^{Created on 2021-09-18 by the [reprex package](https://reprex.tidyverse.org) (v2.0.1)}

Thank you,

Toufiq

Answer 1

[Gordon Smyth]

Simply add the counts from the technical replicates, for example by using the sumTechReps function in the edgeR package, which is provided for this purpose. Taking means is not appropriate.

[mohammedtoufiq91]

Gordon Smyth Noted. Thank you very much.