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Dear experts,

Recently, I have read your important article. I am trying to follow step by step. However,
I cannot generate any data with the command:
python /home/nanobiology/miniconda3/bin/nanopolish_makerange.py \ ~/workspace/your_organism_name.contigs.fasta
| parallel –results nanopolish.results -P 2
nanopolish variants
--consensus polished.{1}.fa -w {1}
--reads ~/workspace/barcodeXX_final_input.fastq
--bam ~/workspace/alignment_sorted.bam
--genome ~/workspace/your_organism_name.contigs.fasta
-t 1

After running this command:

python /home/leo/bin/anaconda3//bin/nanopolish_makerange.py /home/leo/BC01_5out_2/BC01_5.contigs.fasta | parallel --results /home/leo/BC01_5_nanopolish/nanopolish.results -P 2 nanopolish variants --consensus –o polished.{1}.fa -w {1} --reads /home/leo/BC01_5_basecalled_finalinput.fastq.index --bam /home/leo/BC01_5_basecalled_alignment_sorted.bam --genome /home/leo/BC01_5out_2/BC01_5.contigs.fasta -t 8

It only produced the nanopolish.results folder, it did not produced the "polished" folder. Therefore I cannot do the next step.
Here is the output results:

Usage: nanopolish variants [OPTIONS] --reads reads.fa --bam alignments.bam --genome genome.fa
Find SNPs using a signal-level HMM

-v, --verbose display verbose output
--version display version
--help display this help and exit
--snps only call SNPs
--consensus run in consensus calling mode
--fix-homopolymers run the experimental homopolymer caller
--faster minimize compute time while slightly reducing consensus accuracy
-w, --window=STR find variants in window STR (format: <chromsome_name>:-)
-r, --reads=FILE the ONT reads are in fasta FILE
-b, --bam=FILE the reads aligned to the reference genome are in bam FILE
-e, --event-bam=FILE the events aligned to the reference genome are in bam FILE
-g, --genome=FILE the reference genome is in FILE
-p, --ploidy=NUM the ploidy level of the sequenced genome
-q --methylation-aware=STR turn on methylation aware polishing and test motifs given in STR (example: -q dcm,dam)
--genotype=FILE call genotypes for the variants in the vcf FILE
-o, --outfile=FILE write result to FILE [default: stdout]
-t, --threads=NUM use NUM threads (default: 1)
-m, --min-candidate-frequency=F extract candidate variants from the aligned reads when the variant frequency is at least F (default 0.2)
-i, --indel-bias=F bias HMM transition parameters to favor insertions (F<1) or deletions (F>1).
this value is automatically set depending on --consensus, but can be manually set if spurious indels are called
-d, --min-candidate-depth=D extract candidate variants from the aligned reads when the depth is at least D (default: 20)
-x, --max-haplotypes=N consider at most N haplotype combinations (default: 1000)
--min-flanking-sequence=N distance from alignment end to calculate variants (default: 30)
--max-rounds=N perform N rounds of consensus sequence improvement (default: 50)
-c, --candidates=VCF read variant candidates from VCF, rather than discovering them from aligned reads
--read-group=RG only use alignments with read group tag RG
-a, --alternative-basecalls-bam=FILE if an alternative basecaller was used that does not output event annotations
then use basecalled sequences from FILE. The signal-level events will still be taken from the -b bam.
--calculate-all-support when making a call, also calculate the support of the 3 other possible bases
--models-fofn=FILE read alternative k-mer models from FILE

Report bugs to https://github.com/jts/nanopolish/issues

variants: too many arguments

Could you help me out of this issue. Addtionally, I read the instructions of nanopolish variants, it required the the "reads.fa" file not the "reads.fastq" file. Is it possible the reason ? If I want to find the reads.fa, how can I find the data in the output?

Thank you very much,

Le Phuong.