I am currently using PRONAME to analyse full-length 16S rRNA Nanopore sequencing data.
I used the gzipped FASTQ (.fastq.gz) files directly from MinKNOW as the input for PRONAME and followed the tutorial step by step. The pipeline completed successfully, and I was able to generate the final output.
However, I would like to perform additional quality control on the reads that passed the proname_filter step. Specifically, I would like to retrieve or export the filtered reads as .fastq or .fastq.gz files so that I can analyse them using FastQC.
Is there a way to obtain the FASTQ files containing only the reads retained after the proname_filter step? If so, could someone please point me to the relevant output directory or command?
Yes, the reads retained after the proname_filter step are already available as FASTQ files, so you do not need to export them separately.
proname_filter creates an HQ/ directory in your PRONAME working directory. Depending on the value used for --datatype, you will find HQ/HQ_simplex_seqs.fastq, HQ/HQ_duplex_seqs.fastq or HQ/HQ_simplex_duplex_seqs.fastq.
Note that PRONAME itself also generates a length-versus-quality scatter plot from these filtered reads by default, which can be useful for visually checking the quality distribution after filtering.
Finally, please do not hesitate to post any PRONAME-specific questions directly on the PRONAME GitHub repository, where it will be easier for us to follow and address issues related to the pipeline.
I located the HQ/HQ_simplex_seqs.fastq file, but it aggregates reads across all multiplexed samples into a single combined file. Consequently, running FastQC/MultiQC evaluates the dataset as a whole rather than per sample.
Is there a built-in parameter or flag in PRONAME to generate separate, filtered FASTQ files for each individual sample/barcode? If not, could you suggest the best way within the pipeline workflow (or via an intermediate directory/log) to demultiplex these filtered reads back into per-sample FASTQ files?