Hey guys....... 
I am new to bioinformatics and have been trying to set up a pipeline for RNA-Seq data analysis. I am currently using a combination of tools, including FastQC for quality control, Trimmomatic for trimming, and STAR for alignment. I have been running into some issues when integrating the tools together, particularly with getting STAR to correctly read the trimmed output from Trimmomatic.
When I run STAR with my trimmed fastq files, I keep getting errors related to input file formatting, even though the same files pass quality checks after trimming. I have tried tweaking the parameters, but nothing seems to fix it. I am also not entirely sure if the problem lies with how I’m specifying the path to the genome index or if something else is off.
I also check this: https://community.france-bioinformatique.fr/t/an-issue-with-the-tool-xcmd-findchrompeaks-mergerokta But I have not found any solution. Could anyone guide me about this? Has anyone else encountered similar problems? If so, I’d really appreciate any advice on troubleshooting this step. Also, if there are any best practices for setting up a seamless pipeline from raw reads to alignment for RNA-Seq, I’d be glad to hear them!
Thank you in advance!
Respected community member! 
Hello,
I'm not able to help you but I advise you to give the exact error message (the output).
Maybe you can also have a look at nf-core pipelines (a community effort to collect a curated set of open‑source analysis pipelines built using Nextflow): Pipelines (for example: rnaseq: Introduction).
Have a good day.
It sounds like you're facing a common issue when setting up RNA-Seq pipelines, particularly with ensuring proper input formatting and correct tool integration. For the STAR alignment issue, it’s essential to verify that your trimmed FASTQ files are properly formatted (e.g., paired-end files must be specified correctly in the STAR command, and the read groups must be consistent). Double-check the paths to both your genome index and the trimmed FASTQ files. If you're using paired-end reads, ensure both files are correctly specified in STAR. Also, ensure you're using the correct trimming parameters with Trimmomatic to avoid any unexpected modifications to the FASTQ headers. If the problem persists, you might want to rerun the trimming process and inspect the output files using head to confirm no unintended changes were made. As for best practices, it’s important to use a consistent workflow, including quality control (FastQC), trimming (Trimmomatic), alignment (STAR), and post-alignment analysis tools, all while ensuring correct file formats and paths.