Error in reference building

[ucbtsh6]

Hello I have a problem in reference building

![Uploading Screen Shot 2020-09-19 at 18.45.31.png…]()

[dg520]

Hi @ucbtsh6 ,

It looks like you failed in the step calculating the mappability. The first error I saw on your screenshot is write error: Disk quota exceeded. Are you sure you have enough disk space?

Can you provide me more information: what is your IRFinder version? Which genome are you working on? And you other dependencies version information please? And please attach here your entire standard error and standard output on you screen after you called IRFinder, instead of a truncated screen shot. I cannot help without these details.

Best, Dadi

[ucbtsh6]

Hi sir,

Thanks for your kind reply. I’m using IRFinder 1.3 version. I’m using killifish genome which I have the GTF and Fasta file for it. And below is the full message I got

Regards, Shaimaa Launching reference build process. The full build might take hours. <Phase 1: STAR Reference Preparation> Sep 19 18:15:15 ..... started STAR run Sep 19 18:15:15 ... starting to generate Genome files Sep 19 18:18:35 ... starting to sort Suffix Array. This may take a long time... Sep 19 18:18:43 ... sorting Suffix Array chunks and saving them to disk... Sep 19 18:21:24 ... loading chunks from disk, packing SA... Sep 19 18:21:49 ... finished generating suffix array Sep 19 18:21:49 ... generating Suffix Array index Sep 19 18:23:41 ... completed Suffix Array index Sep 19 18:23:41 ..... processing annotations GTF Sep 19 18:24:26 ..... inserting junctions into the genome indices Sep 19 18:26:23 ... writing Genome to disk ... Sep 19 18:26:31 ... writing Suffix Array to disk ... Sep 19 18:26:46 ... writing SAindex to disk Sep 19 18:26:50 ..... finished successfully <Phase 2: Mapability Calculation> Sep 19 18:26:52 ... mapping genome fragments back to genome... Sep 19 18:34:53 ... sorting aligned genome fragments... [W::sam_read1] Parse error at line 24079786 samtools sort: truncated file. Aborting Sep 19 18:35:15 ... indexing aligned genome fragments... samtools index: "genome_fragments.bam" is in a format that cannot be usefully indexed Sep 19 18:35:15 ... filtering aligned genome fragments by chromosome/scaffold... [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for indexed BAM or CRAM files. [main_samview] random alignment retrieval only works for

[dg520]

Hi @ucbtsh6 ,

Which version of Samtools are your using? Please also let me know the versions of other IRFinder dependencies. And how much memory do you have one your machine? How does your command line to call IRFinder look like? Was it called directly? Or was this submitted to a queuing system? If via a queuing system, how many CPU threads and memory sizes did you allocate? Please provide all the above information.

You error came here: [W::sam_read1] Parse error at line 24079786. samtools sort: truncated file. Aborting during Phase 2. It failed to sort the aligned SAM file. Multiple factors could cause this, including but not limited to:

1. Your alignment to genome is not complete
2. Your Samtools is incompatible
3. Your disk status is limited to hold all temporary files.

To further debug your issue, I need you to download IRFinder 1.3.1 And re-run your commands above. In 1.3.0, all temporary files will be cleaned. But they will be kept in 1.3.1, so that you can investigate the files right at the error step.

After you re-run the command in 1.3.1, I believe you would fail around the same place. You need to cd into your IRFinder reference folder. Then cd into Mapability folder, where you should see genome_fragments.sam file. Try to run the following:

samtools sort -@ 2 genome_fragments.sam > genome_fragments.bam

Can this complete successfully? If not, please attach here all the standard output and standard errors on your screen.

Best, Dadi

[ucbtsh6]

Hello, I changed the place to have more space and I got the message bellow. The samtools I’m using is samtools 1.9 I’m not doing it on my machine I’m doing it in UCL server by submitting to a queuing system.

This Is the job I submit

! /bin/bash

$ -S /bin/bash

$ -l tmem=36G

$ -l h_vmem=36G

$ -j y

$ -N newestIRFinerfromstarref

$ -o /SAN/bahlerlab/killifish_transcriptome/shaimaa/chiku.result/

$ -e /SAN/bahlerlab/killifish_transcriptome/shaimaa/chiku.result/errors

$ -wd /SAN/bahlerlab/killifish_transcriptome/shaimaa/IRFinder-1.3.0/

echo "Starting IRFinder"

IRFinder -m BuildRefProcess -r REF/genome\ -e REF/extra-input-files/RNA.SpikeIn.ERCC.fasta.gz

echo "Finish job"

This is the message I got

Launching reference build process. The full build might take hours. <Phase 1: STAR Reference Preparation> Sep 20 02:16:47 ..... started STAR run Sep 20 02:16:47 ... starting to generate Genome files Sep 20 02:18:53 ... starting to sort Suffix Array. This may take a long time... Sep 20 02:19:02 ... sorting Suffix Array chunks and saving them to disk... Sep 20 02:21:39 ... loading chunks from disk, packing SA... Sep 20 02:22:07 ... finished generating suffix array Sep 20 02:22:07 ... generating Suffix Array index Sep 20 02:23:48 ... completed Suffix Array index Sep 20 02:23:48 ..... processing annotations GTF Sep 20 02:24:18 ..... inserting junctions into the genome indices Sep 20 02:26:07 ... writing Genome to disk ... Sep 20 02:26:13 ... writing Suffix Array to disk ... Sep 20 02:26:37 ... writing SAindex to disk Sep 20 02:26:41 ..... finished successfully <Phase 2: Mapability Calculation> Sep 20 02:26:42 ... mapping genome fragments back to genome... Sep 20 02:34:28 ... sorting aligned genome fragments... [bam_sort_core] merging from 16 files and 16 in-memory blocks... Sep 20 02:36:13 ... indexing aligned genome fragments... Sep 20 02:36:20 ... filtering aligned genome fragments by chromosome/scaffold... Sep 20 02:39:36 ... merging filtered genome fragments... Sep 20 02:40:18 ... calculating regions for exclusion... Sep 20 02:42:28 ... cleaning temporary files... <Phase 3: IRFinder Reference Preparation> Sep 20 02:42:36 ... building Ref 1... Sep 20 02:42:52 ... building Ref 2... Sep 20 02:42:54 ... building Ref 3... Sep 20 02:42:54 ... building Ref 4... Sep 20 02:43:00 ... building Ref 5... Sep 20 02:43:07 ... building Ref 6... Sep 20 02:43:07 ... building Ref 7... Sep 20 02:43:07 ... building Ref 8... Sep 20 02:43:07 ... building Ref 9... Sep 20 02:43:07 ... building Ref 10c... Sep 20 02:43:07 ... building Ref 11c... Error: introns.unique.bed is empty. Error: ref-cover.bed is empty. Error: ref-read-continues.ref is empty. Error: ref-sj.ref is empty. Error: IRFinder reference building FAILED. Finish job

Many thanks for your help Shaimaa On 20 Sep 2020, at 02:32, Dadi notifications@github.com<mailto:notifications@github.com> wrote:

Hi @ucbtsh6https://github.com/ucbtsh6 ,

Which version of Samtools are your using? Please also let me know the versions of other IRFinder dependencies. And how much memory do you have one your machine? How does your command line to call IRFinder look like? Was it called directly? Or was this submitted to a queuing system? If via a queuing system, how many CPU threads and memory sizes did you allocate? Please provide all the above information.

You error came here: [W::sam_read1] Parse error at line 24079786. samtools sort: truncated file. Aborting during Phase 2. It failed to sort the aligned SAM file. Multiple factors could cause this, including but not limited to:

1. Your alignment to genome is not complete
2. Your Samtools is incompatible
3. Your disk status is limited to hold all temporary files. To further debug your issue, I need you to download IRFinder 1.3.1 And re-run your commands above. In 1.3.0, all temporary files will be cleaned. But they will be kept in 1.3.1, so that you can investigate the files right at the error step.

After you re-run the command in 1.3.1, I believe you would fail around the same place. You need to cd into your IRFinder reference folder. Then cd into Mapability folder, where you should see genome_fragments.sam file. Try to run the following:

samtools sort -@ 2 genome_fragments.sam > genome_fragments.bam

Can this complete successfully? If not, please attach here all the standard output and standard errors on your screen.

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/williamritchie/IRFinder/issues/118#issuecomment-695448007, or unsubscribehttps://github.com/notifications/unsubscribe-auth/ANVUPRM63YJW7D3BSSPD7B3SGVLRTANCNFSM4RTERD6Q.

[dg520]

Hi @ucbtsh6 ,

Great. Changing disk space has obviously solved your previous issue that led to the mapability calculation failure. Please double-check in your IRFinder reference folder, the sub-folder Mapability should now contain a gzipped file. Let me know if it is not the case.

Now you still have empty reference generated, which is highly likely due to your GTF structure. Open your GTF/GFF/GFF3 file, whichever you used for IRFinder reference preparation. That file must contain the following tags. It also must contain lines marked at exon (at the 3rd column). If not, you have to do it manually. These are the typical Ensembl GTF/GFF/GFF3 file structure that might be disrupted on a user-specific genome.

Best, Dadi

[ucbtsh6]

Hi Dadi, Thank you so much for your help. So I found this file in the Mapability subfolder MapabilityExclusion.bed.gz

And my GTF file is look like that So there is no gene_name, nor transcript and gene_biotype. I can add the gene name column but I don’t know from where I can get the transcript or gene_biotype? [cid:9691A68F-02D6-4E93-A19A-A3DE8717E8B3@ucl.ac.uk] Regards, Shaimaa On 20 Sep 2020, at 16:55, Dadi notifications@github.com<mailto:notifications@github.com> wrote:

Hi @ucbtsh6https://github.com/ucbtsh6 ,

Great. Changing disk space has obviously solved your previous issue that led to the mapability calculation failure. Please double-check in your IRFinder reference folder, the sub-folder Mapability should now contain a gzipped file. Let me know if it is not the case.

Now you still have empty reference generated, which is highly likely due to your GTF structure. Open your GTF/GFF/GFF3 file, whichever you used for IRFinder reference preparation. That file must contain the following tagshttps://github.com/williamritchie/IRFinder/wiki/Build-Reference#using-customized-gtf. It also must contain lines marked at exon (at the 3rd column). If not, you have to do it manually. These are the typical Ensembl GTF/GFF/GFF3 file structure that might be disrupted on a user-specific genome.

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/williamritchie/IRFinder/issues/118#issuecomment-695802852, or unsubscribehttps://github.com/notifications/unsubscribe-auth/ANVUPRLUTBA5EIKQFYOPCZLSGYQXLANCNFSM4RTERD6Q.

[dg520]

Hi @ucbtsh6 .

Lacking of transcript or gene_biotype will definitely lead to empty reference. The precise labeling of them requires the understanding of the genome, which is out of my scope. If you can get it from somewhere, that'll be great.

However, to maximize your searching space for intron retention, you can ARTIFICIALLY mark all your gene/transcript as "protein_coding" or "processed_transcript". At the moment, this might be the best practise to answer your downstream biological question.

Please also refer to a human genome GTF on Ensembl FTP site, to learn how to mark each tag in a correct format. IRFinder is very sensitive to the format.

Best, Dadi

[ucbtsh6]

Hi Dadi, I changed GTF and add what is required [cid:97796C32-DC8F-4CED-8571-0A303D0F10C4@ucl.ac.uk] And I still get the same error. What could I do? Starting IRFinder Launching reference build process. The full build might take hours. <Phase 1: STAR Reference Preparation> Sep 21 20:47:03 ..... started STAR run Sep 21 20:47:03 ... starting to generate Genome files Sep 21 20:48:52 ... starting to sort Suffix Array. This may take a long time... Sep 21 20:49:01 ... sorting Suffix Array chunks and saving them to disk... Sep 21 20:52:05 ... loading chunks from disk, packing SA... Sep 21 20:53:52 ... finished generating suffix array Sep 21 20:53:52 ... generating Suffix Array index Sep 21 20:55:51 ... completed Suffix Array index Sep 21 20:55:51 ..... processing annotations GTF Sep 21 20:56:17 ..... inserting junctions into the genome indices Sep 21 20:57:56 ... writing Genome to disk ... Sep 21 20:58:01 ... writing Suffix Array to disk ... Sep 21 20:58:25 ... writing SAindex to disk Sep 21 20:58:30 ..... finished successfully <Phase 2: Mapability Calculation> Sep 21 20:58:30 ... mapping genome fragments back to genome... Sep 21 21:06:21 ... sorting aligned genome fragments... [bam_sort_core] merging from 16 files and 16 in-memory blocks... Sep 21 21:08:02 ... indexing aligned genome fragments... Sep 21 21:08:09 ... filtering aligned genome fragments by chromosome/scaffold... Sep 21 21:10:54 ... merging filtered genome fragments... Sep 21 21:11:29 ... calculating regions for exclusion... Sep 21 21:13:39 ... cleaning temporary files... <Phase 3: IRFinder Reference Preparation> Sep 21 21:13:48 ... building Ref 1... Sep 21 21:14:01 ... building Ref 2... Sep 21 21:14:03 ... building Ref 3... Sep 21 21:14:03 ... building Ref 4... Sep 21 21:14:08 ... building Ref 5... Sep 21 21:14:14 ... building Ref 6... Sep 21 21:14:14 ... building Ref 7... Sep 21 21:14:14 ... building Ref 8... Sep 21 21:14:14 ... building Ref 9... Sep 21 21:14:14 ... building Ref 10c... Sep 21 21:14:14 ... building Ref 11c... Error: introns.unique.bed is empty. Error: ref-cover.bed is empty. Error: ref-read-continues.ref is empty. Error: ref-sj.ref is empty. Error: IRFinder reference building FAILED. Finish job

Regards Shaimaa

On 20 Sep 2020, at 18:11, Dadi notifications@github.com<mailto:notifications@github.com> wrote:

Hi @ucbtsh6https://github.com/ucbtsh6 .

Lacking of transcript or gene_biotype will definitely lead to empty reference. The precise labeling of them requires the understanding of the genome, which is out of my scope. If you can get it from somewhere, that'll be great.

However, to maximize your searching space for intron retention, you can ARTIFICIALLY mark all your gene/transcript as "protein_coding" or "processed_transcript". At the moment, this might be the best practise to answer your downstream biological question.

Please also refer to a human genome GTF on Ensembl FTP site, to learn how to mark each tag in a correct format. IRFinder is very sensitive to the format.

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/williamritchie/IRFinder/issues/118#issuecomment-695811510, or unsubscribehttps://github.com/notifications/unsubscribe-auth/ANVUPRKF5NB47PQVDAEGFYLSGYZU3ANCNFSM4RTERD6Q.

[dg520]

Hi @ucbtsh6 ,

In your IRFinder reference folder, there is a sub-folder called IRFinder. I think you should now see multiple files starting with tmp. Among them, does tmp.candidate.introns exist but is empty? If so, you still didn't get the GTF right. If it's not empty or if it doesn't exist, let me know.

Best, Dadi

[ucbtsh6]

Hi Dadi, You are right. I modified my GTF file and now it seems to work properly and didn’t give any error in reference building. As below, Launching reference build process. The full build might take hours. <Phase 1: STAR Reference Preparation> Sep 22 09:03:03 ..... started STAR run Sep 22 09:03:03 ... starting to generate Genome files Sep 22 09:05:04 ... starting to sort Suffix Array. This may take a long time... Sep 22 09:05:13 ... sorting Suffix Array chunks and saving them to disk... Sep 22 09:07:49 ... loading chunks from disk, packing SA... Sep 22 09:08:45 ... finished generating suffix array Sep 22 09:08:45 ... generating Suffix Array index Sep 22 09:10:36 ... completed Suffix Array index Sep 22 09:10:36 ..... processing annotations GTF Sep 22 09:11:02 ..... inserting junctions into the genome indices Sep 22 09:12:35 ... writing Genome to disk ... Sep 22 09:12:44 ... writing Suffix Array to disk ... Sep 22 09:13:12 ... writing SAindex to disk Sep 22 09:13:15 ..... finished successfully <Phase 2: Mapability Calculation> Sep 22 09:13:16 ... mapping genome fragments back to genome... Sep 22 09:20:57 ... sorting aligned genome fragments... [bam_sort_core] merging from 16 files and 16 in-memory blocks... Sep 22 09:22:31 ... indexing aligned genome fragments... Sep 22 09:22:38 ... filtering aligned genome fragments by chromosome/scaffold... Sep 22 09:25:42 ... merging filtered genome fragments... Sep 22 09:26:26 ... calculating regions for exclusion... Sep 22 09:28:40 ... cleaning temporary files... <Phase 3: IRFinder Reference Preparation> Sep 22 09:28:48 ... building Ref 1... Sep 22 09:29:04 ... building Ref 2... Sep 22 09:29:07 ... building Ref 3... Sep 22 09:29:07 ... building Ref 4... Sep 22 09:29:22 ... building Ref 5... Sep 22 09:29:38 ... building Ref 6... Sep 22 09:29:39 ... building Ref 7... Sep 22 09:29:40 ... building Ref 8... Sep 22 09:29:41 ... building Ref 9... Sep 22 09:29:41 ... building Ref 10c... Sep 22 09:29:41 ... building Ref 11c... Finish job However, when I try to do IR quantification it showed this error Argument error: -r REF/genome/, Does not appear to be a valid IRFinder reference. Could not find REF/genome//IRFinder/ref-cover.bed

Could you please help me with this Regards Shaimaa

On 21 Sep 2020, at 21:42, Dadi notifications@github.com<mailto:notifications@github.com> wrote:

Hi @ucbtsh6https://github.com/ucbtsh6 ,

In your IRFinder reference folder, there is a sub-folder called IRFinder. I think you should now see multiple files starting with tmp. Among them, does tmp.candidate.introns exist but is empty? If so, you still didn't get the GTF right. If it's not empty or if it doesn't exist, let me know.

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/williamritchie/IRFinder/issues/118#issuecomment-696363811, or unsubscribehttps://github.com/notifications/unsubscribe-auth/ANVUPRMREJP2PFEIBXWFTK3SG63FDANCNFSM4RTERD6Q.

[dg520]

Hi @ucbtsh6 ,

It depends on in which folder you submit your IRFinder quantification job to the queuing system, the relative path REF/genome may not work. Can you try to use the absolute path of REF/genome instead?

Best, Dadi

[ucbtsh6]

Hi Dadi, I’m so grateful for your continuous help and support I managed to build the reference and now I’m quantifying the IR regions and I get this message WARN: Very low portion of reads have a splice junction. This may indicate the experiment is not an mRNA-Seq experiment. “END”

Is there anything to worry about ?? Or its okey? Regards, Shaimaa

On 22 Sep 2020, at 16:00, Dadi notifications@github.com<mailto:notifications@github.com> wrote:

Hi @ucbtsh6https://github.com/ucbtsh6 ,

It depends on in which folder you submit your IRFinder quantification job to the queuing system, the relative path REF/genome may not work. Can you try to use the absolute path of REF/genome instead?

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/williamritchie/IRFinder/issues/118#issuecomment-696778891, or unsubscribehttps://github.com/notifications/unsubscribe-auth/ANVUPRL5SZ43U7VYKLYFQ4LSHC3ZXANCNFSM4RTERD6Q.

[dg520]

Hi @ucbtsh6 ,

I'm glad the pipeline finally worked on your side.

The warning means the number of spliced reads in your RNASeq is lowered than "expected". The observed value is caculated as a proportion of your total splicing reads against the total number of splice junctions in your genome annotation. The expected value is arbitary set by us internally. For a typical RNASeq, this warning should not appear.

As I'm not familiar with your species of interest, here is my best suggestions but you have to ask yourself:

1. Is your splice junction annotation robust (e.g. low false positive rate, the junction coordinates are precise, etc)?
2. Did you use the GTF that matches your FASTA? A mismatch would let the GTF assign junctions at very wrong locus.
3. Did you sequence a tissue that only expresses a few gene at low level?
4. Is this a transcriptome-wide RNASeq or did you pre-select a set of genes before sequencing?
5. Do you think your RNASeq is successful (e.g. no DNA contamination)? You can investigate your BAM file for things like what proportion of reads are uniquely mapped? What proportion of reads uniquely mapped to gene regions? Are they consistent with other's RNASeq experiment on the same species?

Best, Dadi

[ucbtsh6]

Hello, Thanks a lot for your help. It fixed now with modifying my GTF file. However, now the problem raise with doing the DESeq analysis part I got this message metaList <- DESeqDataSetFromIRFinder(filePaths=paths, designMatrix=experiment) Error in scan(file = file, what = what, sep = sep, quote = quote, dec = dec, : line 6 did not have 21 elements Called from: scan(file = file, what = what, sep = sep, quote = quote, dec = dec, nmax = nrows, skip = 0, na.strings = na.strings, quiet = TRUE, fill = fill, strip.white = strip.white, blank.lines.skip = blank.lines.skip, multi.line = FALSE, comment.char = comment.char, allowEscapes = allowEscapes, flush = flush, encoding = encoding, skipNul = skipNul) Browse[1]> Could you suggest anything to help? Regards Shaimaa On 23 Sep 2020, at 17:02, Dadi notifications@github.com<mailto:notifications@github.com> wrote:

Hi @ucbtsh6https://github.com/ucbtsh6 ,

I'm glad the pipeline finally worked on your side.

The warning means the number of spliced reads in your RNASeq is lowered than "expected". The observed value is caculated as a proportion of your total splicing reads against the total number of splice junctions in your genome annotation. The expected value is arbitary set by us internally. For a typical RNASeq, this warning should not appear.

As I'm not familiar with your species of interest, here is my best suggestions but you have to ask yourself:

1. Is your splice junction annotation robust (e.g. low false positive rate, the junction coordinates are precise, etc)?
2. Did you use the GTF that matches your FASTA? A mismatch would let the GTF assign junctions at very wrong locus.
3. Did you sequence a tissue that only expresses a few gene at low level?
4. Is this a transcriptome-wide RNASeq or did you pre-select a set of genes before sequencing?
5. Do you think your RNASeq is successful (e.g. no DNA contamination)? You can investigate your BAM file for things like what proportion of reads are uniquely mapped? What proportion of reads uniquely mapped to gene regions? Are they consistent with other's RNASeq experiment on the same species?

Best, Dadi

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/williamritchie/IRFinder/issues/118#issuecomment-697600488, or unsubscribehttps://github.com/notifications/unsubscribe-auth/ANVUPRPG2RIFKX7FAFY7GM3SHILZ3ANCNFSM4RTERD6Q.

[dg520]

Hi @ucbtsh6 ,

Can you manually print out the variable paths on your screen in an R console and check whether each path is valid (e.g. each path links to an existing file and that file is an IRFinder output)? And as I said before, I suggest you use absolute file paths to make both paths and experiment variables. Relative file paths won't work if you submitted your job from a wrong directory.

Best, Dadi