Manual

Modules available:

nanopolish extract: extract reads in FASTA or FASTQ format from a directory of FAST5 files
nanopolish call-methylation: predict genomic bases that may be methylated
nanopolish variants: detect SNPs and indels with respect to a reference genome
nanopolish variants --consensus: calculate an improved consensus sequence for a draft genome assembly
nanopolish eventalign: align signal-level events to k-mers of a reference genome
nanopolish phase-reads: Phase reads using heterozygous SNVs with respect to a reference genome
nanopolish polya: Estimate polyadenylated tail lengths on native RNA reads

extract

Overview

This module is used to extract reads in FASTA or FASTQ format from a directory of FAST5 files.

Input

• path to a directory of FAST5 files modified to contain basecall information

Output

• sequences of reads in FASTA or FASTQ format

Usage example

nanopolish extract [OPTIONS] <fast5|dir>

Argument name(s)	Required	Default value	Description
<fast5|dir>	Y	NA	FAST5 or path to directory of FAST5 files.
-r, --recurse	N	NA	Recurse into subdirectories
-q, --fastq	N	fasta format	Use when you want to extract to FASTQ format
-t, --type=TYPE	N	2d-or-template	The type of read either: {template, complement, 2d, 2d-or-template, any}
-b, --basecaller=NAME[:VERSION]	N	NA	consider only data produced by basecaller NAME, optionally with given exact VERSION
-o, --output=FILE	N	stdout	Write output to FILE

index

Overview

Build an index mapping from basecalled reads to the signals measured by the sequencer

Input

• path to directory of raw nanopore sequencing data in FAST5 format

• basecalled reads

Output

• gzipped FASTA file of basecalled reads (.index)

• index files (.fai, .gzi, .readdb)

Readdb file format

Readdb file is a tab-separated file that contains two columns. One column represents read ids and the other column represents the corresponding path to FAST5 file:

read_id_1   /path/to/fast5/containing/reads_id_1/signals
read_id_2   /path/to/fast5/containing/read_id_2/signals

Usage example

nanopolish index [OPTIONS] -d nanopore_raw_file_directory reads.fastq

Argument name(s)	Required	Default value	Description
-d, --directory	Y	NA	FAST5 or path to directory of FAST5 files containing ONT sequencing raw signal information.
-f, --fast5-fofn	N	NA	file containing the paths to each fast5 for the run

call-methylation

Overview

Classify nucleotides as methylated or not.

Input

• Basecalled ONT reads in FASTA format

Output

• tab-separated file containing per-read log-likelihood ratios

Usage example

nanopolish call-methylation [OPTIONS] <fast5|dir>

Argument name(s)	Required	Default value	Description
-r, --reads=FILE	Y	NA	the ONT reads are in fasta FILE
-b, --bam=FILE	Y	NA	the reads aligned to the genome assembly are in bam FILE
-g, --genome=FILE	Y	NA	the genome we are computing a consensus for is in FILE
-t, --threads=NUM	N	1	use NUM threads
--progress	N	NA	print out a progress message

variants

Overview

This module is used to call single nucleotide polymorphisms (SNPs) using a signal-level HMM.

Input

• basecalled reads

• alignment info

• genome assembly

Output

• VCF file

Usage example

nanopolish variants [OPTIONS] --reads reads.fa --bam alignments.bam --genome genome.fa

Argument name(s)	Required	Default value	Description
--snps	N	NA	use flag to only call SNPs
--consensus=FILE	N	NA	run in consensus calling mode and write polished sequence to FILE
--fix-homopolymers	N	NA	use flag to run the experimental homopolymer caller
--faster	N	NA	minimize compute time while slightly reducing consensus accuracy
-w, --window=STR	N	NA	find variants in window STR (format: <chromsome_name>:<start>-<end>)
-r, --reads=FILE	Y	NA	the ONT reads are in fasta FILE
-b, --bam=FILE	Y	NA	the reads aligned to the reference genome are in bam FILE
-e, --event-bam=FILE	Y	NA	the events aligned to the reference genome are in bam FILE
-g, --genome=FILE	Y	NA	the reference genome is in FILE
-o, --outfile=FILE	N	stdout	write result to FILE
-t, --threads=NUM	N	1	use NUM threads
-m, --min-candidate-frequency=F	N	0.2	extract candidate variants from the aligned reads when the variant frequency is at least F
-d, --min-candidate-depth=D	N	20	extract candidate variants from the aligned reads when the depth is at least D
-x, --max-haplotypes=N	N	1000	consider at most N haplotypes combinations
--max-rounds=N	N	50	perform N rounds of consensus sequence improvement
-c, --candidates=VCF	N	NA	read variants candidates from VCF, rather than discovering them from aligned reads
-a, --alternative-basecalls-bam=FILE	N	NA	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	N	NA	when making a call, also calculate the support of the 3 other possible bases
--models-fofn=FILE	N	NA	read alternatives k-mer models from FILE

event align

Overview

Align nanopore events to reference k-mers

Input

• basecalled reads

• alignment information

• assembled genome

Usage example

nanopolish eventalign [OPTIONS] --reads reads.fa --bam alignments.bam --genome genome.fa

Argument name(s)	Required	Default value	Description
--sam	N	NA	use to write output in SAM format
-w, --window=STR	N	NA	Compute the consensus for window STR (format : ctg:start_id-end_id)
-r, --reads=FILE	Y	NA	the ONT reads are in fasta FILE
-b, --bam=FILE	Y	NA	the reads aligned to the genome assembly are in bam FILE
-g, --genome=FILE	Y	NA	the genome we are computing a consensus for is in FILE
-t, --threads=NUM	N	1	use NUM threads
--scale-events	N	NA	scale events to the model, rather than vice-versa
--progress	N	NA	print out a progress message
-n, --print-read-names	N	NA	print read names instead of indexes
--summary=FILE	N	NA	summarize the alignment of each read/strand in FILE
--samples	N	NA	write the raw samples for the event to the tsv output
--models-fofn=FILE	N	NA	read alternative k-mer models from FILE

phase-reads - (experimental)

Overview

Phase reads using heterozygous SNVs with respect to a reference genome

Input

• basecalled reads

• alignment information

• assembled genome

• variants (from nanopolish variants or from other sources eg. Illumina VCF)

Usage example

nanopolish phase-reads [OPTIONS] --reads reads.fa --bam alignments.bam --genome genome.fa variants.vcf

polya

Overview

Estimate the number of nucleotides in the poly(A) tails of native RNA reads.

Input

• basecalled reads

• alignment information

• reference transcripts

Usage example

nanopolish polya [OPTIONS] --reads=reads.fa --bam=alignments.bam --genome=ref.fa

Argument name(s)	Required	Default value	Description
-w, --window=STR	N	NA	Compute only for reads aligning to window of reference STR (format : ctg:start_id-end_id)
-r, --reads=FILE	Y	NA	the FAST(A/Q) file of native RNA reads
-b, --bam=FILE	Y	NA	the BAM file of alignments between reads and the reference
-g, --genome=FILE	Y	NA	the reference transcripts
-t, --threads=NUM	N	1	use NUM threads
-v, -vv	N	NA	-v returns raw sample log-likelihoods, while -vv returns event durations