GFFParser error with example data in sqanti_qc

[chaesee1]

Hello,

I got error that sqanti_qc.py with example data. the error is as follows:

(SQANTI3.env) [SQANTI3-5.2]$ sqanti3_qc.py example/UHR_chr22.gtf example/gencode.v38.basic_chr22.gtf example/GRCh38.p13_chruman.refTSS_v3.1.hg38.bed --polyA_motif_list data/polyA_motifs/mouse_and_human.polyA_motif.txt -o UHR_chr22 -d example/SQANTI3_QC_output2 -fl exUHR_chr22_short_reads.fofn --cpus 4 --report both
Rscript (R) version 4.3.1 (2023-06-16)
WARNING: output directory /ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQANTI3_QC_output2 already exists. Overwriting!
Write arguments to /ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQANTI3_QC_output2/UHR_chr22.params.txt...
**** Running SQANTI3...
**** Parsing provided files....
Reading genome fasta /ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/GRCh38.p13_chr22.fasta....
Error corrected FASTA /ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQANTI3_QC_output2/UHR_chr22_corrected.fasta already exists. Using it.
**** Predicting ORF sequences...
ORF file /ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQANTI3_QC_output2/UHR_chr22_corrected.faa already exists. Using it....
**** Parsing Reference Transcriptome....
/ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQANTI3_QC_output2/refAnnotation_UHR_chr22.genePred already exists. Using it.
**** Parsing Isoforms....
**** Running STAR for calculating Short-Read Coverage.
START running STAR...
Index identified. Proceeding to mapping.
Mapping for UHR_Rep1_chr22 : in progress...
Mapping for UHR_Rep1_chr22 : done.
/home/denovo/anaconda3/envs/SQANTI3.env/bin/STAR-avx2 --runThreadN 4 --genomeDir /ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQA/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/UHR_Rep1_chr22.R1.fastq.gz example/UHR_Rep1_chr22.R2.fastq.gz --outFileNamePrefix /ess/dlstibm/AQC_output2/STAR_mapping/UHR_Rep1_chr22 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterType BySJout --outSAMunmapped Within --outFilt 0.04 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --sjdbScore 1 --genomeLoad NoSharedMemFilesCommand zcat --twopassMode Basic
STAR version: 2.7.11a compiled: 2023-09-15T02:58:53+0000 :/opt/conda/conda-bld/star_1694746407721/work/source
Jan 22 17:15:03 ..... started STAR run
Jan 22 17:15:03 ..... loading genome
Jan 22 17:15:04 ..... started 1st pass mapping
Jan 22 17:15:46 ..... finished 1st pass mapping
Jan 22 17:15:46 ..... inserting junctions into the genome indices
Jan 22 17:15:55 ..... started mapping
Jan 22 17:16:39 ..... finished mapping
Jan 22 17:16:39 ..... started sorting BAM
Jan 22 17:16:40 ..... finished successfully
Mapping for UHR_Rep2_chr22 : in progress...
Mapping for UHR_Rep2_chr22 : done.
/home/denovo/anaconda3/envs/SQANTI3.env/bin/STAR-avx2 --runThreadN 4 --genomeDir /ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQA/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/UHR_Rep2_chr22.R1.fastq.gz example/UHR_Rep2_chr22.R2.fastq.gz --outFileNamePrefix /ess/dlstibm/AQC_output2/STAR_mapping/UHR_Rep2_chr22 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterType BySJout --outSAMunmapped Within --outFilt 0.04 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --sjdbScore 1 --genomeLoad NoSharedMemFilesCommand zcat --twopassMode Basic
STAR version: 2.7.11a compiled: 2023-09-15T02:58:53+0000 :/opt/conda/conda-bld/star_1694746407721/work/source
Jan 22 17:16:40 ..... started STAR run
Jan 22 17:16:40 ..... loading genome
Jan 22 17:16:41 ..... started 1st pass mapping
Jan 22 17:17:15 ..... finished 1st pass mapping
Jan 22 17:17:15 ..... inserting junctions into the genome indices
Jan 22 17:17:24 ..... started mapping
Jan 22 17:17:57 ..... finished mapping
Jan 22 17:17:57 ..... started sorting BAM
Jan 22 17:17:57 ..... finished successfully
Input pattern: /ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQANTI3_QC_output2/STAR_mapping/.
The following files found and to be read as junctions:
/ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQANTI3_QC_output2/STAR_mapping/UHR_Rep2_chr22SJ.out.tab
/ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/example/SQANTI3_QC_output2/STAR_mapping/UHR_Rep1_chr22SJ.out.tab
6762 junctions read. 2 junctions added to both strands because no strand information from STAR.
Running calculation of TSS ratio
Traceback (most recent call last):
File "/ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/sqanti3_qc.py", line 2542, in
main()
File "/ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/sqanti3_qc.py", line 2525, in main
run(args)
File "/ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/sqanti3_qc.py", line 1888, in run
isoforms_info, ratio_TSS_dict = isoformClassification(args, isoforms_by_chr, refs_1exon_by_chr, refs_exons_by_chr, junctions_by_chr, junctions_by_gene, start_ends_by_gene, genome_dict, indelsJunc, orfDict, corrGTF)
File "/ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/sqanti3_qc.py", line 1558, in isoformClassification
inside_bed, outside_bed = get_TSS_bed(corrGTF, chr_order)
File "/ess/dlstibm/Application/IsoSeq/SQANTI3-5.2/utilities/short_reads.py", line 122, in get_TSS_bed
for rec in BCBio_GFF.parse(in_handle, limit_info=limit_info, target_lines=1):
File "/home/denovo/anaconda3/envs/SQANTI3.env/lib/python3.10/site-packages/BCBio/GFF/GFFParser.py", line 793, in parse
for rec in parser.parse_in_parts(gff_files, base_dict, limit_info,
File "/home/denovo/anaconda3/envs/SQANTI3.env/lib/python3.10/site-packages/BCBio/GFF/GFFParser.py", line 337, in parse_in_parts
cur_dict = self._results_to_features(cur_dict, results)
File "/home/denovo/anaconda3/envs/SQANTI3.env/lib/python3.10/site-packages/BCBio/GFF/GFFParser.py", line 376, in _results_to_features
base = self._add_parent_child_features(base, results.get('parent', []),
File "/home/denovo/anaconda3/envs/SQANTI3.env/lib/python3.10/site-packages/BCBio/GFF/GFFParser.py", line 448, in _add_parent_child_features
child_feature = self._get_feature(child_dict)
File "/home/denovo/anaconda3/envs/SQANTI3.env/lib/python3.10/site-packages/BCBio/GFF/GFFParser.py", line 591, in _get_feature
new_feature = SeqFeature.SeqFeature(location, feature_dict['type'],
TypeError: SeqFeature.init() got an unexpected keyword argument 'strand'

Thanks,

• chaehwa.

[xiuru]

I encountered the same issue. Did you manage to resolve it?

[SamGallaher]

This appears to be an issue with the GFFparser.py file in BCBio and compatibility with biopython- see here
They suggest a workaround by installing biopython<=1.81