Thank you very much for the reply and sorry about the confusion.
1. Replications: Actually, I am working on multiple publicly available RNA datasets from several sulfate-reducing bacteria for machine learning studies. I can grasp your clarification about replications and this problem is resolved. (Question resolved)
2. Hisat2 error:: I am working on a separate RNA seq-dataset from our wet-lab experiment with triplicates. Please find the link below for the history named “RNA seq Gr Day 60”. Here we have RNA seq data of Desulfovibrio alaskensis bacteria exposed to five different experimental conditions. Below are the GALAXY pipeline along with their file numbers. Also, as I mentioned in my previous email, I see that I have an issue with the HISAT2 file. Among the columns in the HISAT2 result, both MPOS and ISIZEcolumns had only zeros for all the genes. When I used the same GALAXY pipeline last month I had several numbers in these columns where I ended up with good results. I am skeptical that may HISAT2 BAM file has some issues and it is reflected in DESEQ2 error. (Question yet to be resolved)
Files 7, 14, 30, 37 and 44 - Raw reads made into a collection of triplicates
143 - Trimmomatic od data collection 7
150 - Trimmomatic od data collection 14
157 - Trimmomatic od data collection 30
164 - Trimmomatic od data collection 37
180 - Trimmomatic od data collection 44 (error)
187 - Reference genome (.fa)
188 - Reference genome (.gff3)
189 - HISAT2 of collection 143
196 - HISAT2 of collection 150
203 - HISAT2 of collection 157
210 - HISAT2 of collection 164
217/218 - Featurecounts for collection 189
231/232 - Featurecounts for collection 196
245/246 - Featurecounts for collection 203
259/260 - Featurecounts for collection 210
273/274 - Deseq 2 on paired-end reads of data 219 (as the collection was showing up while uploading data). Please find attached figures of screenshots.
3. Deseq2 error?: Later I released that I may be running paired-end instead of collections as the collection was not showing up when I upload for Deseq2. Please find attached screenshots of the mouse pointer at Collection and Multiple datasets. (Question yet to be resolved)
I believe problems 2 and 3 might be related. It would be great if you could clarify this.
4. QC and Trimmomatic error for a replicate among triplicates: QC and Trimmomatic showed error for one replicate (the third one) among the triplicate. The raw data were in file 44, the third replicate. Files 129, 130 (QC) and 180 (trimmomatic) were the error files and I didn’t process this sample to the next stage. (Question yet to be resolved)
Thank you very much and please let me know for more information. My research was stuck because of this issue for the past two weeks. It would be great if you could help me resolve this.