open(os.path.join(tempDir, "%s_inconsistent.bed" % chrom),'rb') as fd

[gaobaoqing-picb]

Copy and paste the exact command you tried to run flair correct -q ${i}_mapped_q1.bed12 -f /data/rnomics8/gaobaoqing/tsa-seq_lxq/20220715_lxq/backup/index/t2t_v2.0_hisat2/chm13.draft_v2.0.gene_annotation.gtf -g /data/rnomics8/gaobaoqing/tsa-seq_lxq/20220715_lxq/backup/index/t2t_v2.0minimap2/chm13v2.0.fa --threads 4 --output sample${i}_flair --print_check

How did you install Flair?

5. git cloned the current repository

What happened?

Step 5/5: Correcting Splice Sites: 100%|██████████████████████████| 25/25 [00:22<00:00,  1.10it/s]
Traceback (most recent call last): 100%|██████████████████████████| 25/25 [00:22<00:00,  1.66s/it]
  File "/data/rnomics8/gaobaoqing/tsa-seq_lxq/nanopore/softwares/flair/src/flair/ssCorrect.py", line 423, in <module>
    main()
  File "/data/rnomics8/gaobaoqing/tsa-seq_lxq/nanopore/softwares/flair/src/flair/ssCorrect.py", line 402, in main
    with open(os.path.join(tempDir, "%s_inconsistent.bed" % chrom),'rb') as fd:
FileNotFoundError: [Errno 2] No such file or directory: '/data/rnomics8/gaobaoqing/tsa-seq_lxq/nanopore/20220909/deal_data/mapping/1/6b18a24c-825e-41d2-ba39-caa548c5aad1/chr1_inconsistent.bed'
Correction command did not exit with success status:
/data/rnomics10/gaobaoqing/anaconda3/envs/tsa-seq2/bin/python3 /data/rnomics8/gaobaoqing/tsa-seq_lxq/nanopore/softwares/flair/src/flair/ssCorrect.py -i 1_mapped_q1.bed12 -w 15 -p 4 -o sample_1_flair --progress -f /data/rnomics8/gaobaoqing/tsa-seq_lxq/20220715_lxq/backup/index/t2t_v2.0_minimap2/chm13v2.0.fa --correctStrand -g /data/rnomics8/gaobaoqing/tsa-seq_lxq/20220715_lxq/backup/index/t2t_v2.0_hisat2/chm13.draft_v2.0.gene_annotation.gtf

We know it's ugly but we promise it helps us solve problems faster.

What else do we need to know? Go ahead, every bit helps.

[Jeltje]

It looks like you don't have write access to /data/rnomics8/gaobaoqing/tsa-seq_lxq/nanopore/20220909/deal_data/mapping/1/6b18a24c-825e-41d2-ba39-caa548c5aad1 Can you verify?

[gaobaoqing-picb]

Thank you for your timely answer! I checked that the folder has writable permission. And there are _known_juncs.bed and _temp_reads.bed files in this folder. Is there any other reason？ Thanks a lot!

[Jeltje]

Can you copy and paste a list of everything in that folder?

Also please give the output of

head -n 5 ${i}mapped_q1.bed12 # (of course replace ${i} with a real file)
head -n 5 /data/rnomics8/gaobaoqing/tsa-seq_lxq/20220715_lxq/backup/index/t2t_v2.0_hisat2/chm13.draft_v2.0.gene_annotation.gtf 
head -n 1 /data/rnomics8/gaobaoqing/tsa-seq_lxq/20220715_lxq/backup/index/t2t_v2.0_minimap2/chm13v2.0.fa

[gaobaoqing-picb]

Thank you very much for your reply！The command you suggest runs as follows.

1. head -n 5 ${i}mapped_q1.bed12

chr1 20530 35061 98:1034|23095722-7104-448b-ae62-b9f10975440d;16 13 - 20530 35061 217,95,2 3 557,180,104, 0,7916,14427, chr1 20531 125183 97:952|2014fa35-5e9b-4a83-9218-f8404f2b3bce;16 37 - 20531 125183 217,95,2 3 556,180,74, 0,7915,104578, chr1 43932 191678 95:2650|8d55c271-6973-46b7-8ae3-38eb131cbca0;0 8 + 43932 191678 27,158,119 8 44,243,949,222,189,70,157,417, 0,127794,128472,131762,136264,138678,141569,147329, chr1 77721 78703 80:1208|b45a388b-71ee-44ac-9259-ab71af738c73;16 9 - 77721 78703 217,95,2 1 982, 0, chr1 127610 128575 83:1028|03ac3e55-2a78-4e9c-8a7a-bb08c5e7a8a0;0 59 + 127610 128575 27,158,119 1 965, 0,

2. head -n 5 /data/rnomics8/gaobaoqing/tsa-seq_lxq/20220715_lxq/backup/index/t2t_v2.0_hisat2/chm13.draft_v2.0.gene_annotation.gtf

chr1 Liftoff transcript 11136 12457 . - . transcript_id "LOFF_T0000001"; gene_id "LOFF_G0000001"; gene_name "AL627309.3"; chr1 Liftoff exon 11136 11635 . - . transcript_id "LOFF_T0000001"; gene_id "LOFF_G0000001"; gene_name "AL627309.3"; chr1 Liftoff exon 11639 12457 . - . transcript_id "LOFF_T0000001"; gene_id "LOFF_G0000001"; gene_name "AL627309.3"; chr1 Liftoff transcript 11630 13433 . + . transcript_id "LOFF_T0000002"; gene_id "LOFF_G0000002"; gene_name "AP006222.2"; chr1 Liftoff exon 11630 11831 . + . transcript_id "LOFF_T0000002"; gene_id "LOFF_G0000002"; gene_name "AP006222.2";

3.head -n 1 /data/rnomics8/gaobaoqing/tsa-seq_lxq/20220715_lxq/backup/index/t2t_v2.0_minimap2/chm13v2.0.fa

chr1 CP068277.2 Homo sapiens isolate CHM13 chromosome 1

[gaobaoqing-picb]

And the list of everything in that folder is as follows.

ls /data/rnomics8/gaobaoqing/tsa-seq_lxq/nanopore/20220909/deal_data/mapping/1/6b18a24c-825e-41d2-ba39-caa548c5aad1

chr10_known_juncs.bed chr15_known_juncs.bed chr1_known_juncs.bed chr3_known_juncs.bed chr8_known_juncs.bed chr10_temp_reads.bed chr15_temp_reads.bed chr1_temp_reads.bed chr3_temp_reads.bed chr8_temp_reads.bed chr11_known_juncs.bed chr16_known_juncs.bed chr20_known_juncs.bed chr4_known_juncs.bed chr9_known_juncs.bed chr11_temp_reads.bed chr16_temp_reads.bed chr20_temp_reads.bed chr4_temp_reads.bed chr9_temp_reads.bed chr12_known_juncs.bed chr17_known_juncs.bed chr21_known_juncs.bed chr5_known_juncs.bed chrM_known_juncs.bed chr12_temp_reads.bed chr17_temp_reads.bed chr21_temp_reads.bed chr5_temp_reads.bed chrM_temp_reads.bed chr13_known_juncs.bed chr18_known_juncs.bed chr22_known_juncs.bed chr6_known_juncs.bed chrX_known_juncs.bed chr13_temp_reads.bed chr18_temp_reads.bed chr22_temp_reads.bed chr6_temp_reads.bed chrX_temp_reads.bed chr14_known_juncs.bed chr19_known_juncs.bed chr2_known_juncs.bed chr7_known_juncs.bed chrY_known_juncs.bed chr14_temp_reads.bed chr19_temp_reads.bed chr2_temp_reads.bed chr7_temp_reads.bed chrY_temp_reads.bed

[Jeltje]

flair calls ssCorrect.py, which calls ssPrep.py, and it looks like that program didn't do what it's supposed to.

Can you try running it separately? The base command is this: ssPrep.py -i chr20_temp_reads.bed -j chr20_known_juncs.bed -o chr20 -f test_ref/genome.fa -w 15 --correctStrand

If you run it from your output directory /data/rnomics8/gaobaoqing/tsa-seq_lxq/nanopore/20220909/deal_data/mapping/1/6b18a24c-825e-41d2-ba39-caa548c5aad1

it should be /data/rnomics10/gaobaoqing/anaconda3/envs/tsa-seq2/bin/python3 /data/rnomics8/gaobaoqing/tsa-seq_lxq/nanopore/softwares/flair/src/flair/ssPrep.py -i chr20_temp_reads.bed -j chr20_known_juncs.bed -o chr20 -f /data/rnomics8/gaobaoqing/tsa-seq_lxq/20220715_lxq/backup/index/t2t_v2.0_minimap2/chm13v2.0.fa -w 15 --correctStrand What happens? Do you get chr20_inconsistent.bed and chr20_corrected.bed?

Also please attach the ssCorrect.err file that should be in your output directory, or paste it in a seprarate comment.

[gaobaoqing-picb]

Thanks a lot! The problem has been solved！ When I run ssPrep.py separately, a new error- "No module named 'ncls'" has appeared. After run "pip install ncls", "flair correct" can run successfully!