Assembly with flye
Flye is a de novo assembler for single-molecule sequencing reads, such as those produced by PacBio and Oxford Nanopore Technologies. It is designed for a wide range of datasets, from small bacterial projects to large mammalian-scale assemblies. The package represents a complete pipeline: it takes raw PacBio / ONT reads as input and outputs polished contigs. Flye also has a special mode for metagenome assembly. Get a usage message of canu on how to use the assembler:
flye --help
usage: flye (--pacbio-raw | --pacbio-corr | --pacbio-hifi | --nano-raw |
--nano-corr | --nano-hq ) file1 [file_2 ...]
--out-dir PATH
[--genome-size SIZE] [--threads int] [--iterations int]
[--meta] [--polish-target] [--min-overlap SIZE]
[--keep-haplotypes] [--debug] [--version] [--help]
[--scaffold] [--resume] [--resume-from] [--stop-after]
[--read-error float] [--extra-params]
[--deterministic]
Assembly of long reads with repeat graphs
options:
-h, --help show this help message and exit
--pacbio-raw path [path ...]
PacBio regular CLR reads (<20% error)
--pacbio-corr path [path ...]
PacBio reads that were corrected with other methods (<3% error)
--pacbio-hifi path [path ...]
PacBio HiFi reads (<1% error)
--nano-raw path [path ...]
ONT reads with odler chemistries, pre R9 Guppy5 (10-20% error)
--nano-corr path [path ...]
ONT reads that were corrected with other methods (<3% error)
--nano-hq path [path ...]
ONT R10 reads, aka Q20 (<3% error). For R9 Guppy5+, increase --read-error slightly
--subassemblies path [path ...]
[deprecated] high-quality contigs input
-g size, --genome-size size
estimated genome size (for example, 5m or 2.6g)
-o path, --out-dir path
Output directory
-t int, --threads int
number of parallel threads [1]
-i int, --iterations int
number of polishing iterations [1]
-m int, --min-overlap int
minimum overlap between reads [auto]
--asm-coverage int reduced coverage for initial disjointig assembly [not set]
--hifi-error float [deprecated] same as --read-error
--read-error float adjust parameters for given read error rate (as fraction e.g. 0.03)
--extra-params extra_params
extra configuration parameters list (comma-separated)
--plasmids unused (retained for backward compatibility)
--meta metagenome / uneven coverage mode
--keep-haplotypes do not collapse alternative haplotypes
--no-alt-contigs do not output contigs representing alternative haplotypes
--scaffold enable scaffolding using graph [disabled by default]
--trestle [deprecated] enable Trestle [disabled by default]
--polish-target path run polisher on the target sequence
--resume resume from the last completed stage
--resume-from stage_name
resume from a custom stage
--stop-after stage_name
stop after the specified stage completed
--debug enable debug output
-v, --version show program's version number and exit
--deterministic perform disjointig assembly single-threaded
Input reads can be in FASTA or FASTQ format, uncompressed
or compressed with gz. Currently, PacBio (CLR, HiFi, corrected)
and ONT reads (regular, HQ, corrected) are supported. Expected error rates are
<15% for PB CLR/regular ONT; <3% for ONT R10, <3% for corrected, and <1% for HiFi. Note that Flye
was primarily developed to run on uncorrected reads. You may specify multiple
files with reads (separated by spaces). Mixing different read
types is not yet supported. The --meta option enables the mode
for metagenome/uneven coverage assembly.
To reduce memory consumption for large genome assemblies,
you can use a subset of the longest reads for initial disjointig
assembly by specifying --asm-coverage and --genome-size options. Typically,
40x coverage is enough to produce good disjointigs.
You can run Flye polisher as a standalone tool using
--polish-target option.
We will run the assembly on the small dataset, to save time. The assembly for the complete dataset will take about one hour. We will perform the assembly as follows:
flye --nano-raw basecall_small/basecall.fastq.gz --out-dir assembly_small --threads 14 --genome-size 2m
After that is done, inspect the results. We can get a quick view on the number of generated contigs with:
grep '>' ~/workdir/assembly_small/assembly.fasta
If there is time, we start the actual assembly with all data now:
flye --nano-raw basecall/basecall.fastq.gz --out-dir assembly --threads 14 --genome-size 2m
Otherwise, copy the precomputed assembly with the complete dataset into your working directory:
cp -r ~/workdir/results/assembly_new/ ~/workdir/
and have a quick look on the number of contigs:
grep '>' ~/workdir/assembly/assembly.fasta