Hi @Sonenshine Thanks for explaining, and great screenshots will all the details, very helpful!
Loading read data should always use default settings. If you don’t get the expected datatype, then this nearly always indicates some content problem to address.
- Getting Data into Galaxy
- Hands-on: NGS data logistics / NGS data logistics / Introduction to Galaxy Analyses
The technical issue is how the + quality score lines are annotated: they are in a legacy Illumina format, and the quality scores themselves may have a legacy Illumina scaling. This has major scientific implications as well. Why? The wrong scaling (from what tools are expecting to process) will throw off all other statistical calculations.
All tools but a few tools will expect Sanger Phred +33, designated as fastqsanger and fastqsanger.gz in Galaxy.
You can load up other formats, and make adjustments (tools will be used to standardize the quality score + annotation line, then re-scale the quality scores themselves). I wrote up some FAQs back when this was a more common necessity, and those should all still work if you want to try. Warning: a bit complicated!
- Let the Upload tool detect the format
- Adjust the + lines first (this should repair the FastQC issue)
- Then “groom” if needed
- See here for all → Galaxy FAQs.
- And, there was another recent discussion about this if you want more details about the “why” and exact steps. → Faster Download and Extract Reads in FASTQ and ENA reads are slightly different - #2 by jennaj
Or, you can get the SRR reads from NCBI already in fastqsanger format. These will already have the + annotation line standardized, and data points rescaled (if needed based on the original sequencing protocol), directly from the archives, with either of these two tools:
- Into collection folders with → Faster Download and Extract Reads in FASTQ format from NCBI SRA
- Into individual datasets with → Download and Extract Reads in FASTQ format from NCBI SRA
Please give those a review, thanks! 