Where can I find the BAM file where reads are associated with bead barcodes and/or molecular barcodes?

The Seeker pipeline does not generate this BAM file , where reads are associated with bead or molecular barcodes , by default . However, the association can be generated by merging two of the pipeline’s intermediate outputs ( r1-db  and  r2-db ). The output will be a parquet file where each row is a read - read ID and bead barcode sequences are defined for each row. Merging of  r1-db  and  r2-db [...]

Why is the FORMATCLEANUP step running out of memory and failing with an error?

This is usually the result of very large fastq.gz files. Very large fastq.gz files that have over 2 – 3 billion reads and/or have sequence lengths much longer than 55 bp can cause the Seeker pipeline to run out of memory when the hardware has limited RAM (256 GB or less). Reduce the barcode_chunk_size parameter in the Seeker Primary Analysis P ipeline. The default barcode_chunk_size default is [...]

How do I decrease or increase the UMI threshold used to remove bead barcodes?

The use of -- bb_min flag during Seeker pipeline execution may be utilized to lower the default UMI cutoff of 10. Here is an example of the Seeker pipeline execution command with the UMI cutoff set to seven (this overrides the default of 10): nextflow run main.nf \ --input /path/to/samplesheet.csv \ --outdir ${root_output_dir}/results/ \ -work-dir ${root_output_dir}/work/ \ [...]

I am encountering the following error when executing the Seeker Primary Analysis Pipeline. What’s wrong?

NOTE: Process `NF:CURIOSEEKER:PRIMARY_READ1:CURIOSEEKER_CALCULATETOPBB_COMBINED (Mouse_spleen_1M)` terminated with an error exit status (255) -- Execution is retried (1) NOTE: Process `NF:CURIOSEEKER:PRIMARY_READ1:CURIOSEEKER_CALCULATETOPBB_COMBINED (Mouse_spleen_1M)` terminated with an error exit status (255) -- Error is ignored Join mismatch for the following entries: key=[id:Mouse_spleen_1M, [...]

We do not have internet access on our Linux server. When executing Seeker v3.0 pipeline with Singularity, the pipeline is unable to download containers for subread, SAMtools, and STAR. This usually results in NextFlow throwing a “Join mismatch” type error. How do I solve this?

Errors Observed: nextflow.exception.AbortOperationException: Join mismatch for the following entries… Follow the instructions below to solve this issue: Download the following file and uncompress it: $ wget https://curioseekerbioinformatics.s3.us-west-1.amazonaws.com/containerImages/curioseeker-public-singularity-dec-2024.tar.gz" target="_blank [...]