nanopolish variants: too many arguments

[123chenshixin]

Hi, I'm using nanopolish variants to polish the draft genome which is the assbemly result by using canu.I use the Illumina sequencing data to polish my draft genome which is named JYF80.contigs.fasta.Since I am the first time using nanopolish, I use the command you have given on the Internet.The command are as follows:

!/bin/bash

for a in JYF80 do draf_fa=/home/cxs3_z4/csx/20201209/canu/${a}/${a}.contigs.fasta read_1_fa=/home/cxs3_z4/cff/Illumina/QC/${a}_R1.fq.gz read_2_fa=/home/cxs3_z4/cff/Illumina/QC/${a}_R2.fq.gz genome=./${a}.contigs.fasta

ln -s ${draf_fa} ./

Index the draft genome

bwa index ${genome}

Align the basecalled reads to the draft sequence

bwa mem -x ont2d -t 8 ${genome} ${read_1_fa} ${read_2_fa} | samtools sort -o reads.sorted.bam -T reads.tmp - samtools index reads.sorted.bam

python3 /home/cxs3_z4/software/nanopolish/scripts/nanopolish_makerange.py ${genome} | parallel --results nanopolish.results -P 8 nanopolish variants --consensus polished.{1}.fa -w {1} -r ${read_1_fa} ${read_2_fa} -b reads.sorted.bam -g ${genome} -t 4 --min-candidate-frequency 0.1 done

Then,the reads.sorted.bam and reads.sorted.bam.bai files are generated.But it tells me that "variants: too many arguments" and print many times of the informations which are the same results by using the command "nanopolish variants -h". It is obvious that the nanopolish variants has problems but I try many methods and they didn't work.My reference genome is 12M which is a kind of yeast.I don't know wheather it is too small for "-P 8".

[jts]

Hi,

The commands you are using are for an old version of nanopolish. Please see the instructions for the latest versions here:

https://nanopolish.readthedocs.io/en/latest/quickstart_consensus.html

Jared

On Dec 26, 2020, at 10:35 PM, 123chenshixin notifications@github.com wrote:

 Hi, I'm using nanopolish variants to polish the draft genome which is the assbemly result by using canu.I use the Illumina sequencing data to polish my draft genome which is named JYF80.contigs.fasta.Since I am the first time using nanopolish, I use the command you have given on the Internet.The command are as follows:

!/bin/bash

for a in JYF80 do draf_fa=/home/cxs3_z4/csx/20201209/canu/${a}/${a}.contigs.fasta read_1_fa=/home/cxs3_z4/cff/Illumina/QC/${a}_R1.fq.gz read_2_fa=/home/cxs3_z4/cff/Illumina/QC/${a}_R2.fq.gz genome=./${a}.contigs.fasta

ln -s ${draf_fa} ./

Index the draft genome

bwa index ${genome}

Align the basecalled reads to the draft sequence

bwa mem -x ont2d -t 8 ${genome} ${read_1_fa} ${read_2_fa} | samtools sort -o reads.sorted.bam -T reads.tmp - samtools index reads.sorted.bam

python3 /home/cxs3_z4/software/nanopolish/scripts/nanopolish_makerange.py ${genome} | parallel --results nanopolish.results -P 8 nanopolish variants --consensus polished.{1}.fa -w {1} -r ${read_1_fa} ${read_2_fa} -b reads.sorted.bam -g ${genome} -t 4 --min-candidate-frequency 0.1 done

Then,the reads.sorted.bam and reads.sorted.bam.bai files are generated.But it tells me that "variants: too many arguments" and print many times of the informations which are the same results by using the command "nanopolish variants -h". It is obvious that the nanopolish variants has problems but I try many methods and they didn't work.My reference genome is 12M which is a kind of yeast.I don't know wheather it is too small for "-P 8".

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[123chenshixin]

I'm soryy that it doesn't work.The problem is the same.I even try to manual input the command. minimap2 -ax map-ont -t 8 ${genome} ${read_1_fa} ${read_2_fa} | samtools sort -o reads.sorted.bam -T reads.tmp samtools index reads.sorted.bam

nanopolish variants --consensus -o polished.vcf \ -w "tig00000001:200000-202000" \ -r ${read_1_fa} ${read_2_fa} \ -b reads.sorted.bam \ -g ${genome}

[jts]

Can you paste the full error message you received?

On Sat, Dec 26, 2020 at 11:42 PM 123chenshixin notifications@github.com wrote:

I'm soryy that it doesn't work.The problem is the same.I even try to manual input the command. minimap2 -ax map-ont -t 8 ${genome} ${read_1_fa} ${read_2_fa} | samtools sort -o reads.sorted.bam -T reads.tmp samtools index reads.sorted.bam

nanopolish variants --consensus -o polished.vcf -w "tig00000001:200000-202000" -r ${read_1_fa} ${read_2_fa} -b reads.sorted.bam -g ${genome}

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[123chenshixin]

The entire output is follows:

[M::mm_idx_gen::0.4881.01] collected minimizers [M::mm_idx_gen::0.6302.01] sorted minimizers [M::main::0.6302.01] loaded/built the index for 20 target sequence(s) [M::mm_mapopt_update::0.7181.88] mid_occ = 31 [M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 20 [M::mm_idx_stat::0.7741.82] distinct minimizers: 2077969 (95.40% are singletons); average occurrences: 1.116; average spacing: 5.337 [M::worker_pipeline::74.2446.41] mapped 3370249 sequences [M::worker_pipeline::91.8215.26] mapped 2497543 sequences [M::worker_pipeline::163.9785.81] mapped 3370249 sequences [M::worker_pipeline::181.520*5.29] mapped 2497543 sequences [M::main] Version: 2.17-r941 [M::main] CMD: minimap2 -ax map-ont -t 8 ./JYF80.contigs.fasta /home/cxs3_z4/cff/Illumina/QC/JYF80_R1.fq.gz /home/cxs3_z4/cff/Illumina/QC/JYF80_R2.fq.gz [M::main] Real time: 182.086 sec; CPU: 961.384 sec; Peak RSS: 3.813 GB [bam_sort_core] merging from 6 files and 1 in-memory blocks... variants: too many arguments

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: :-) -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

[jts]

Oh, I see the problem now. You are trying to use nanopolish with paired end illumina data. Nanopolish only supports nanopore data.

Jared

On Dec 27, 2020, at 12:15 AM, 123chenshixin notifications@github.com wrote:

 The entire output is follows:

[M::mm_idx_gen::0.4881.01] collected minimizers [M::mm_idx_gen::0.6302.01] sorted minimizers [M::main::0.6302.01] loaded/built the index for 20 target sequence(s) [M::mm_mapopt_update::0.7181.88] mid_occ = 31 [M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 20 [M::mm_idx_stat::0.7741.82] distinct minimizers: 2077969 (95.40% are singletons); average occurrences: 1.116; average spacing: 5.337 [M::worker_pipeline::74.2446.41] mapped 3370249 sequences [M::worker_pipeline::91.8215.26] mapped 2497543 sequences [M::worker_pipeline::163.9785.81] mapped 3370249 sequences [M::worker_pipeline::181.520*5.29] mapped 2497543 sequences [M::main] Version: 2.17-r941 [M::main] CMD: minimap2 -ax map-ont -t 8 ./JYF80.contigs.fasta /home/cxs3_z4/cff/Illumina/QC/JYF80_R1.fq.gz /home/cxs3_z4/cff/Illumina/QC/JYF80_R2.fq.gz [M::main] Real time: 182.086 sec; CPU: 961.384 sec; Peak RSS: 3.813 GB [bam_sort_core] merging from 6 files and 1 in-memory blocks... variants: too many arguments

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: :-) -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

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[123chenshixin]

But when I use nanopore sequences to polish my draft genome, it semms that it must require the fast5 file which is used to build the index files for nanopore sequences. However, I don't have the fast5 file for some reasons.Wheather I can run nanopolish without it? To confirm the error,the results of running the command from the website you have given are as follows:

Error: no fast5 files found [M::mm_idx_gen::0.5780.99] collected minimizers [M::mm_idx_gen::0.7831.71] sorted minimizers [M::main::0.7831.71] loaded/built the index for 20 target sequence(s) [M::mm_mapopt_update::0.8681.64] mid_occ = 31 [M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 20 [M::mm_idx_stat::0.9201.60] distinct minimizers: 2079913 (95.37% are singletons); average occurrences: 1.117; average spacing: 5.331 [M::worker_pipeline::81.4797.71] mapped 61731 sequences [M::worker_pipeline::126.2886.75] mapped 56266 sequences [M::worker_pipeline::128.1126.66] mapped 21087 sequences [M::main] Version: 2.17-r941 [M::main] CMD: minimap2 -ax map-ont -t 8 ./JYF80.racon3.fasta /home/cxs3_z4/yeast/nanopore/ONT_JYF80.fastq [M::main] Real time: 128.209 sec; CPU: 853.525 sec; Peak RSS: 5.408 GB [bam_sort_core] merging from 2 files and 1 in-memory blocks... [fai_load] build FASTA index. error: could not load the index files for input file /home/cxs3_z4/yeast/nanopore/ONT_JYF80.fastq Please run nanopolish index on your reads (see documentation) [vcf2fasta] rewrote contig tig00000001 with 0 subs, 0 ins, 0 dels (0 skipped) [vcf2fasta] rewrote contig tig00000002 with 0 subs, 0 ins, 0 dels (0 skipped) .........

[jts]

Sorry but you need the fast5s for nanopolish.

Jared

On Sun, Dec 27, 2020 at 9:40 PM 123chenshixin notifications@github.com wrote:

But when I use nanopore sequences to polish my draft genome, it semms that it must require the fast5 file which is used to build the index files for nanopore sequences. However, I don't have the fast5 file for some reasons.Wheather I can run nanopolish without it? To confirm the error,the results of running the command from the website you have given are as follows:

Error: no fast5 files found [M::mm_idx_gen::0.578 0.99] collected minimizers [M::mm_idx_gen::0.7831.71] sorted minimizers [M::main::0.783 1.71] loaded/built the index for 20 target sequence(s) [M::mm_mapopt_update::0.8681.64] mid_occ = 31 [M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 20 [M::mm_idx_stat::0.920 1.60] distinct minimizers: 2079913 (95.37% are singletons); average occurrences: 1.117; average spacing: 5.331 [M::worker_pipeline::81.4797.71] mapped 61731 sequences [M::worker_pipeline::126.288 6.75] mapped 56266 sequences [M::worker_pipeline::128.1126.66] mapped 21087 sequences [M::main] Version: 2.17-r941 [M::main] CMD: minimap2 -ax map-ont -t 8 ./JYF80.racon3.fasta /home/cxs3_z4/yeast/nanopore/ONT_JYF80.fastq [M::main] Real time: 128.209 sec; CPU: 853.525 sec; Peak RSS: 5.408 GB [bam_sort_core] merging from 2 files and 1 in-memory blocks... [fai_load] build FASTA index. error: could not load the index files for input file /home/cxs3_z4/yeast/nanopore/ONT_JYF80.fastq Please run nanopolish index on your reads (see documentation) [vcf2fasta] rewrote contig tig00000001 with 0 subs, 0 ins, 0 dels (0 skipped) [vcf2fasta] rewrote contig tig00000002 with 0 subs, 0 ins, 0 dels (0 skipped) .........

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[123chenshixin]

OK. I will try in another way. Thanks for repplying my questions!