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yuntianf / Longcell-pre

Preprocessing for single cell/Visium Nanopore long reads sequencing to generate isoform quantification
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something warning when using the gtf2bed step #3

Open yuanwsy opened 4 months ago

yuanwsy commented 4 months ago

Hi,

Thanks for developing this very useful and much needed tool!

When using the step1: transform gtf to gene bed,before finished,the command line give a warning: Warning message: In .get_cds_IDX(mcols0$type, mcols0$phase) : The "phase" metadata column contains non-NA values for features of type stop_codon. This information was ignored.

Dose it matter when I using the following step?

yuntianf commented 4 months ago

Hi, It shouldn't matter the downstream step. If you want a double check you could share part of the output. And we notice that LongcellPre is a wrap of bash scripts, which is kind hard to use, currently we have rebuilt it into an R package, and will release it in recent two weeks. I would recommend to use that new version.

Thanks

yuanwsy commented 4 months ago

the output contains three type of file ,

the first file is exon_gtf.rds with five column,the first line looks like: 3143476 | 3144545 | ENSMUSG00000102 | ENSMUST00000193812.2 | ENSMUSE00001343744.2

the second file is gene_bed.rds with six column,the first line looks like: chr1 | 3143476 | 3144545 | 1070 | + | ENSMUSG00000102

and there is alot of file ,like ENSMUSG00000031.txt ,looks like: chr1 60382482 60382616 135 +

is it right ?

Looking forward to see the R package qucikly！ Thanks for developing such a good tool!

yuanwsy commented 4 months ago

and it seems matter?

when I ran the next step ,it cannot generate useful output,all the output is 0kb it comes alot error like:

Namespace(bam='/data/R02/yuanwsy/Longcell-pre/OEalignment/OEalignVM34.bam', gene_bed='/data/R02/yuanwsy/Longcell-pre/mousebed/ENSMUSG00000112.txt', out_path='/data/R02/yuanwsy/Longcell-pre/OEisoquantificationVm34//exon_reads/', toolkit='3') Traceback (most recent call last): File "./SoftclipsExon/softclip_splicesite.py", line 166, in for samrd in sam_input.fetch(gene_chr,gene_start, gene_end): File "pysam/libcalignmentfile.pyx", line 1091, in pysam.libcalignmentfile.AlignmentFile.fetch File "pysam/libchtslib.pyx", line 688, in pysam.libchtslib.HTSFile.parse_region ValueError: invalid coordinates: start (138382074) > stop (81240539) cat: /data/R02/yuanwsy/Longcell-pre/OEisoquantificationVm34//exon_reads/EN*: No such file or directory Extraction of softclips and splice sites finished! Namespace(barcodes='/data/R02/yuanwsy/Longcell-pre/OEbarcodes.txt.txt', batch=100, cores=16, cos_thresh=0.25, kmer=8, mu=20, output='/data/R02/yuanwsy/Longcell-pre/OEisoquantificationVm34//barcode_match/bc.txt', project='Longcell_bc', seq='/data/R02/yuanwsy/Longcell-pre/OEisoquantificationVm34//softclips/softclips.txt', sigma=10, sigma_start=10, top=8) tasks will run on 16 cores sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied sh: /data/R02/yuanwsy/Longcell-pre/scripts/BarcodeMatch/BarcodeMatch: Permission denied Traceback (most recent call last): File "./BarcodeMatch/BarcodeMatch.py", line 107, in barcode_match(args) File "./BarcodeMatch/BarcodeMatch.py", line 98, in barcode_match for line in open(filename): FileNotFoundError: [Errno 2] No such file or directory: 'Longcell_bc_output_sub_0.txt' Barcode Match finished!

yuntianf commented 4 months ago

Hi yuanwsy, Thanks for the updating from @alihamraoui for the gtf annotation for mouse. You could try it again to see if it could work. If it still runs into errors, because this version of Longcell-pre would not be maintained, please wait a while for the new package, I will try my best to get it out this week. Thanks