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No rotated contigs found #67

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Yali1989 opened this issue Dec 5, 2023 · 2 comments
Open

No rotated contigs found #67

Yali1989 opened this issue Dec 5, 2023 · 2 comments

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@Yali1989
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Yali1989 commented Dec 5, 2023

Hi，
Thank you for the useful program. I am running this program with the test data. Every steps was successful except circularization. I got the error message.
2023-12-05 17:20:41 [INFO] Welcome to MitoHifi v2. Starting pipeline...
2023-12-05 17:20:42 [INFO] Length of related mitogenome is: 15372 bp
2023-12-05 17:20:42 [INFO] Number of genes on related mitogenome: 37
2023-12-05 17:20:42 [INFO] Running MitoHifi pipeline in reads mode...
2023-12-05 17:20:42 [INFO] 1. First we map your Pacbio HiFi reads to the close-related mitogenome
2023-12-05 17:20:42 [INFO] minimap2 -t 4 --secondary=no -ax map-hifi test_output/NC_079697.1.fasta ilDeiPorc1.reads.100.fa | /public/home/ynuzjl_lee4/software/samtools-1.18/samtools view -@ 4 -b -F4 -F 0x800 -o reads.HiFiMapped.bam
2023-12-05 17:20:42 [INFO] 2. Now we filter out any mapped reads that are larger than the reference mitogenome to avoid NUMTS
2023-12-05 17:20:42 [INFO] 2.1 First we convert the mapped reads from BAM to FASTA format:
2023-12-05 17:20:42 [INFO] /public/home/ynuzjl_lee4/software/samtools-1.18/samtools fasta reads.HiFiMapped.bam > gbk.HiFiMapped.bam.fasta
2023-12-05 17:20:42 [INFO] Total number of mapped reads: 97
2023-12-05 17:20:42 [INFO] 2.2 Then we filter reads that are larger than 15372 bp
2023-12-05 17:20:42 [INFO] Number of filtered reads: 97
2023-12-05 17:20:42 [INFO] 3. Now let's run hifiasm to assemble the mapped and filtered reads!
2023-12-05 17:20:42 [INFO] hifiasm --primary -t 4 -f 0 -o gbk.HiFiMapped.bam.filtered.assembled gbk.HiFiMapped.bam.filtered.fasta
cat: gbk.HiFiMapped.bam.filtered.assembled.a_ctg.fa: No such file or directory
2023-12-05 17:20:42 [INFO] 4. Let's run the blast of the contigs versus the close-related mitogenome
2023-12-05 17:20:42 [INFO] 4.1. Creating BLAST database:
2023-12-05 17:20:42 [INFO] /public/home/ynuzjl_lee4/software/ncbi-blast-2.15.0+/bin/makeblastdb -in test_output/NC_079697.1.fasta -dbtype nucl
2023-12-05 17:20:45 [INFO] Makeblastdb done.
2023-12-05 17:20:45 [INFO] 4.2. Running blast of contigs against close-related mitogenome:
2023-12-05 17:20:45 [INFO] /public/home/ynuzjl_lee4/software/ncbi-blast-2.15.0+/bin/blastn -query hifiasm.contigs.fasta -db test_output/NC_079697.1.fasta -num_threads 4 -out contigs.blastn -outfmt 6 std qlen slen
2023-12-05 17:20:48 [INFO] Blast done.
2023-12-05 17:20:48 [INFO] 5. Filtering BLAST output to select target sequences
2023-12-05 17:20:48 [INFO] Filtering thresholds applied:
2023-12-05 17:20:48 [INFO] Minimum query percentage = 50
2023-12-05 17:20:48 [INFO] Minimum query length = 80% subject length
2023-12-05 17:20:48 [INFO] Maximum query length = 5 times subject length
2023-12-05 17:20:48 [INFO] Filtering BLAST finished. A list of the filtered contigs was saved on ./contigs_filtering/contigs_ids.txt file
2023-12-05 17:20:48 [INFO] 6. Now we are going to circularize, annotate and rotate each filtered contig. Those are potential mitogenome(s).
2023-12-05 17:20:48 [INFO] Annotation will be done using MitoFinder (default)
2023-12-05 17:20:48 [INFO] Working with contig ptg000001l
2023-12-05 17:20:48 [INFO] Started ptg000001l circularization
/public/home/ynuzjl_lee4/miniconda3/lib/python3.7/site-packages/Bio/SearchIO/_legacy/NCBIStandalone.py:45: BiopythonWarning: Parsing BLAST plain text output file is not a well supported functionality anymore. Consider generating your BLAST output for parsing as XML or tabular format instead.
BiopythonWarning
2023-12-05 17:20:56 [INFO] ptg000001l circularization done. Circularization info saved on ./potential_contigs/ptg000001l/ptg000001l.circularisationCheck.txt
2023-12-05 17:20:56 [INFO] Started ptg000001l (MitoFinder) annotation
2023-12-05 17:24:19 [INFO] ptg000001l annotation done. Annotation log saved on ./potential_contigs/ptg000001l/ptg000001l.annotation_MitoFinder.log
2023-12-05 17:24:19 [INFO] Started ptg000001l rotation.
2023-12-05 17:24:19 [INFO] 7. Now the rotated contigs will be aligned
No rotated contigs found.
An error has possibly occurred during annotation and/or rotation of your contigs

Any idea what can cause the problem, and how to fix the error?
Thanks!
@marcelauliano
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marcelauliano commented Dec 5, 2023 via email

@Yali1989
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Yali1989 commented Dec 6, 2023

Thanks for your reply.
I make local installation on our HPC. I used the following command on test data.
download a reference
python ../src/findMitoReference.py --species "Phalera bucephala" --outfolder test_output --min_length 14000
run MitoHiFi
python ../src/mitohifi.py -c ilPhaBuce1_contig.fa -f ./test_output/OQ830676.1.fasta -g ./test_output/OQ830676.1.gb -t 4 -o 5

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@marcelauliano @Yali1989