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Old 08-04-2016, 02:56 PM   #129
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Location: Boston

Join Date: May 2013
Posts: 2
Default Run Trim Galore! before or after merging technical replicates

I'm quite new to NGS. We just did 4 lanes (2 lanes twice) of Illumina HiSeq Rapid Run 2x51 RNA sequencing of 24 samples. The bcl to fastq conversion was run for us, so every sample has 4 R1 forward fastq files and 4 R2 reverse files. I merged the technical replicates (merged the 4 R1 files, then merged the 4 R2 files) doing a basic command line cat and append. I also ran FastQC on the individual technical replicates, as well as on the merged files. I now plan to upload my files to the Galaxy pipeline for the remainder of the QA/QC and analysis, and was going to start with Trim Galore. But now I'm wondering if Trim Galore needs to work on the original unmerged technical replicates rather than the merged files. E.g., the quality at the beginning of all our reads was spiky, possibly indicating sequencing of the same sequence, and may need to be trimmed; but can trimming the first n bases of each of the 4 files still be done after the files have been merged? So do I upload the unmerged fastq files and run Trim Galore, and then merge them, or upload the merged files and run Trim Galore? Thank you.
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