Open imilenkovic opened 3 years ago
Hey! Thanks for reporting this. It does look like an annotation issue yes. Could you make a sample of your bam available as well so I can debug this? The reference and annotation should be exactly the same as the one used to make the bam files.
Thanks a lot for your reply!
I attached a bam sample (copied the first ~20 rows). bam_sample.txt
Regarding the annotation and the reference files: The annotation is actually a .bed file: https://github.com/ribosomeprofiling/references_for_riboflow/tree/master/transcriptome/mouse/v1/riboflow_annot_and_ref - the one called appris_mouse_v1_actual_regions.bed. The reads were mapped to the transcriptome (but not a single .fa file - bowtie2 was used for mapping so there are 4 .bt2 files).
Let me know if this can still be used or the fastqs will need to be remapped to be compatible with this pipeline.
Thanks a lot!
Ah ok, so, RiboseQC is actually designed to run with genomic alignments, and a gtf file - you'll need to align your reads to the genome rather than the transcriptome. Gencode gtfs should work fine for this.
Hello,
Thanks for clarifying!
I remapped my reads using STAR, and these are the reference and annotation I used:
--genomeFastaFiles ~/references/GRCm38.p6.genome.fa \ --sjdbGTFfile ~/RiboseQC/gencode.vM27.annotation.gtf \
And then I tried running RiboseQC by using the same reference and same annotation (only fasta file converted to 2bit with faToTwoBit):
prepare_annotation_files(annotation_directory = ".", twobit_file = "~/references/GRCm38.p6.genome.2bit", gtf_file = "~/RiboseQC/gencode.vM27.annotation.gtf", scientific_name = "Mus.musculus", annotation_name = "vM27",export_bed_tables_TxDb = F,forge_BSgenome = T,create_TxDb = T)
but I run into the following error:
Creating the TxDb object --- Done! Fri Jul 2 17:56:03 2021 Extracting genomic regions ... Fri Jul 2 17:56:03 2021 Extracting ids and biotypes ... Fri Jul 2 17:58:32 2021 Error in loadFUN(x, seqname, ranges) : trying to load regions beyond the boundaries of non-circular sequence "chr4" Calls: prepare_annotation_files ... loadSubseqsFromStrandedSequence -> loadFUN -> loadFUN In addition: There were 50 or more warnings (use warnings() to see the first 50) Execution halted
Any ideas what might have gone wrong? Thanks!
No problem. That looks to me like your gtf file is for a different version of the genome than the one in the fasta (a range in chr4 is asking for a coordinate that's too high) , I would look at the gencode page to confirm you have the right one.
Hello,
I tried running RiboseQC on bam files obtained with another RiboSeq analysis pipeline called riboflow.
The analysis fails at the P-site step:
Calculating P-sites offsets ... Mon Jun 28 20:39:29 2021 Calculating P-sites offsets --- Done! Mon Jun 28 20:39:29 2021 Calculating P-sites positions and junctions ... Mon Jun 28 20:39:29 2021 Calculating P-sites positions and junctions --- Done! Mon Jun 28 20:40:34 2021 Building aggregate P-sites profiles ... Mon Jun 28 20:40:34 2021 [1] "Not enough signal or low frame preference, skipped P-sites & codon occupancy calculation. \n Set 'all' in the 'choose_readlengths' choice to ignore this warning and get P-sites positions in a new run" Building aggregate P-sites profiles --- Done! Mon Jun 28 20:40:34 2021 Exporting files ... Mon Jun 28 20:40:34 2021 Error in .local(object, con, format, ...) : Scores must be non-NA numeric values Calls: RiboseQC_analysis ... export -> export -> export -> export -> export -> .local In addition: There were 12 warnings (use warnings() to see them) Execution halted
I tried adding the "readlength_choice_method = "all"" parameter in the RiboseQC_analysis, but it gave the same error and it wouldn't export any files.
I am wondering if it has to do with the annotation and reference files I was using for building the bams (https://github.com/ribosomeprofiling/references_for_riboflow/tree/master/transcriptome/mouse/v1/raw)? Or is there something else I might be doing wrong?
Thanks a lot in advance.