SQANTI-reads ERROR: The filtering was too scrict and no genes were found that meet the criteria.

[andysaiz]

Trying to run SQANTI-reads on three samples to test out, but the process does not finish and no report is generated. Ran each through IsoQuant w/ GENCODE reference, SQANTI3 QC, filter, and rescue. Files used for SQANTI-reads input are those generated furthest down the SQANTI3 pipeline (after rescue). I verified that there is an "FL" column in the classification.txt file that has counts. Any suggestions on what to try?

The error message I am getting: ERROR: The filtering was too scrict and no genes were found that meet the criteria.

nohup: ignoring input
Converting GTF to ENSEMBL format
Analyzing chromosomes:
['1', '10', '11', '12', '13', '14', '15', '16', '17', '18', '19', '2', '20', '21', '22', '3', '4', '5', '6', '7', '8', '9', 'M', 'X']
Converting GTF to ENSEMBL format
Analyzing chromosomes:
['1', '10', '11', '11_KI270721v1_random', '12', '13', '14', '15', '16', '17', '17_GL000205v2_random', '18', '19', '2', '20', '21', '22', '22_KI270734v1_random', '3', '4', '5', '6', '7', '8', '9', 'M', 'Un_GL000195v1', 'Un_GL000219v1', 'Un_KI270466v1', 'X', 'Y']
Converting GTF to ENSEMBL format
Analyzing chromosomes:
['1', '10', '11', '11_KI270721v1_random', '12', '13', '14', '14_GL000009v2_random', '15', '16', '17', '17_GL000205v2_random', '18', '19', '2', '20', '21', '22', '22_KI270733v1_random', '22_KI270734v1_random', '3', '4', '5', '6', '7', '8', '9', 'M', 'Un_GL000195v1', 'Un_GL000219v1', 'Un_KI270466v1', 'Un_KI270467v1', 'X', 'Y']
Loading junction file: NT_rep1
Loading classification file: NT_rep1
NT_rep1 done processing
Memory cleared for next sample
Loading junction file: NT_rep2
Loading classification file: NT_rep2
NT_rep2 done processing
Memory cleared for next sample
Loading junction file: NT_rep3
Loading classification file: NT_rep3
NT_rep3 done processing
Memory cleared for next sample
ERROR: The filtering was too scrict and no genes were found that meet the criteria.
[INFO] You inputted SQANTI3 directories, we will run sqanti_reads in fast mode for sample /mnt/data/projects/ebola/MAS_iso_STRAP/sqanti_reads_input/NT_rep1
[INFO] You inputted SQANTI3 directories, we will run sqanti_reads in fast mode for sample /mnt/data/projects/ebola/MAS_iso_STRAP/sqanti_reads_input/NT_rep2
[INFO] You inputted SQANTI3 directories, we will run sqanti_reads in fast mode for sample /mnt/data/projects/ebola/MAS_iso_STRAP/sqanti_reads_input/NT_rep3
**** Calculating UJCs...
**** Calculating UJCs...
**** Calculating UJCs...
/mnt/data/sqanti3/release_sqanti3/sqanti3_reads.py
python /mnt/data/sqanti3/release_sqanti3/src/utilities/sqanti_reads_tables_and_plots_02ndk.py --ref /mnt/data/projects/ebola/MAS_iso_STRAP/gencode.v48.annotation.gtf --design /mnt/data/projects/ebola/MAS_iso_STRAP/sqanti_reads_design_file.csv -o /mnt/data/projects/ebola/MAS_iso_STRAP/sqanti_reads_input --gene-expression 1 --jxn-expression 1 --perc-coverage 10 --perc-junctions 50 --report both --prefix sqantiReads

Input directory is structured like this. All files were generated by running SQANTI3. I renamed some of the files to fit with SQANTI-reads nomenclature requirements:

├── NT_rep1
│   ├── NT_rep1_classification.txt
│   ├── NT_rep1_corrected.gtf
│   └── NT_rep1_junctions.txt
├── NT_rep2
│   ├── NT_rep2_classification.txt
│   ├── NT_rep2_corrected.gtf
│   └── NT_rep2_junctions.txt
└── NT_rep3
├── NT_rep3_classification.txt
├── NT_rep3_corrected.gtf
└── NT_rep3_junctions.txt

Design file looks like this:

sampleID,file_acc
NT_rep1,NT_rep1
NT_rep2,NT_rep2
NT_rep3,NT_rep3

Tried relaxing the filter arguments to be less stringent:

python /mnt/data/sqanti3/release_sqanti3/sqanti3_reads.py \
--genome /mnt/data/projects/ebola/MAS_iso_STRAP/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna \
-i /mnt/data/projects/ebola/MAS_iso_STRAP/sqanti_reads_input \
-de /mnt/data/projects/ebola/MAS_iso_STRAP/sqanti_reads_design_file.csv \
--dir /mnt/data/projects/ebola/MAS_iso_STRAP/sqanti_reads_input \
-t 10 \
-n 8 \
-ge 1 \
-je 1 \
-pc 10 \
-pj 50 \
--report both \
--force_id_ignore \
--annotation /mnt/data/projects/ebola/MAS_iso_STRAP/gencode.v48.annotation.gtf \
--verbose

[pabloati]

Hi, does SQANTI reads just stay frozen at a step or do you get the ERROR of everything filtered out as you mentioned? If it gets frozen, perhaps it is due to the size of your dataset. How many reads do you have in total?

Also, keep in mind that SQANTI3 reads was originally designed to work at read level, rather than at transcript level, to act a quality control for experiments. I have personally never tried it with the results of rescue. However, it should work though.