Problem with with DE_EdgeR

[Former user]

I'm having a problem with the DE_EdgeR source code when I run it in R. I'm looking at miRNAs and using v 1.7.2 from a linux binary on Ubuntu (BioLinux).

I'm able to load everything in and I run QC_EdgeR fine

C_EdgeR(projectdir="/data/pe/miRNA_seq/files/files2/AGRF_CAGRF17481_CCC51ANXX/", dir="/data/pe/miRNA_seq/files/files2/AGRF_CAGRF17481_CCC51ANXX/Readcount_results/", file="nat_bw1-ReadCount.tab", targetfile="/data/software/miARma/cbbio-miarma-42e187a7f514/Examples/basic_examples/miRNAs/data/targets_test1.txt", label="nat_bw1", filter="yes", cpmvalue=2, repthreshold=1, normethod="TMM") Loading required package: edgeR Loading required package: limma Loading required package: ggplot2 Loading required package: gplots

Attaching package: ‘gplots’

The following object is masked from ‘package:stats’:

lowess

Loading required package: genefilter 2018-05-28 13:12:23 Starting quality control analysis of nat_bw1 2018-05-28 13:12:23 The file nat_bw1_QC_Report.pdf has been generated in /data/pe/miRNA_seq/files/files2/AGRF_CAGRF17481_CCC51ANXX/ [1] "/data/pe/miRNA_seq/files/files2/AGRF_CAGRF17481_CCC51ANXX//nat_bw1_QC_Report.pdf"

When I run DE_EdgeR, I however get the following error and I'm not sure why it not recognizing the the targets and contrast files as there are 19 samples in 5 groups.

DE_EdgeR(projectdir="/data/pe/miRNA_seq/files/files2/AGRF_CAGRF17481_CCC51ANXX/", dir="/data/pe/miRNA_seq/files/files2/AGRF_CAGRF17481_CCC51ANXX/Readcount_results/", file="/data/pe/miRNA_seq/files/files2/AGRF_CAGRF17481_CCC51ANXX/Readcount_results/nat_bw1-ReadCount.tab", targetfile="/data/software/miARma/cbbio-miarma-42e187a7f514/Examples/basic_examples/miRNAs/data/targets_test1.txt", contrastfile="/data/software/miARma/cbbio-miarma-42e187a7f514/Examples/basic_examples/miRNAs/data/contrast_test1.txt", label="nat_bw1", filter="yes", cpmvalue=4, repthreshold=2, normethod="TMM", replicates="yes") 2018-05-28 13:12:42 Starting Differential expression analysis with EdgeR of nat_bw1 Error in exactTest(dgenorm, pair = c(contrastval[2], contrastval[1])) : At least one element of given pair is not a group. Groups are: CMSC DMSC LB

I have tried running it using a .ini file but it keeps giving me the resultsfile not found error. The example files runs fine.

Thanks in advance for your help.

[Eduardo Andres Leon]

Hi, For a DE experiment you'll need a group of samples (two at least and three or more are recommended).

As I see in your target file, There is only one CMSC and a DMSC sample.

[Phil Melton]

So I removed those two samples and still gives me the following error,

error in exactTest(dgenorm, pair = c(contrastval[2], contrastval[1])) : At least one element of given pair is not a group. Groups are: LB

There are three other groups - do they have to be equal numbers for each group?

[Eduardo Andres Leon]

Hi, No, you need at least 3 samples in each group. Take care of the format fo the target.txt file, as I see in your error you only have 1 group (LB).

My recommendation: Create a new target and new contrast file just with the samples you want to compare. Then move all sam/bam files not in these two groups from your folder Bowtie1_results (in that way, in this folder you'll see only the files you are going to analyse). Rerun miARma, but only the Readcount and the DEAnalysis sections. Thus, your counts file will contain the samples to be processed according with the target.txt file.

E

[Phil Melton]

In my target file I have 3 groups (LB, LT, and CS) so what I’m confused is why they are not being picked up in Edge R and only stating one group – all with more than 3 samples per group

Filename Name Cat 15248_CCC51ANXX_CCGTCC_L008_R1 15248 LB 16244_CCC51ANXX_GTCCGC_L008_R1 16244 LB 17563_CCC51ANXX_ATGTCA_L008_R1 17563 LB 18487_CCC51ANXX_CTTGTA_L008_R1 18487 CS 18581_CCC51ANXX_TAGCTT_L008_R1 18581 CS 18854_CCC51ANXX_ACTTGA_L008_R1 18854 CS 18855_CCC51ANXX_GATCAG_L008_R1 18855 CS 18858_CCC51ANXX_AGTTCC_L008_R1 18858 LB 18907_CCC51ANXX_AGTCAA_L008_R1 18907 LB 18913_CCC51ANXX_GTAGAG_L008_R1 18913 LB 18955_CCC51ANXX_GCCAAT_L008_R1 18955 LT 18978_CCC51ANXX_ATCACG_L008_R1 18978 LT 18982_CCC51ANXX_ACAGTG_L008_R1 18982 LT 18984_CCC51ANXX_TGACCA_L008_R1 18984 LT 18996_CCC51ANXX_GGCTAC_L008_R1 18996 CS 19003_CCC51ANXX_CAGATC_L008_R1 19003 CS 19033_CCC51ANXX_CGATGT_L008_R1 19033 LT

[Eduardo Andres Leon]

Did you check that all of them are separated by tabs ?

[Phil Melton]

Yep, that fixed it, thanks for your help.

[Eduardo Andres Leon]

A pleasure!