# Create a Genome Assembly

at least 2 cores/threads available
paired-end reads from an Illumina sequencer in FASTQ format gzip recommended
- deconvolved with deconvolve (QuickDeconvolution) or equivalent IMPORTANT

If you have single-sample data, you might be interested in a genome assembly. Unlike metagenome assemblies, a classic genome assembly assumes there is exactly one genome present in your sequences and will try to assemble the most contiguous sequences for this one individual.

usage
harpy metassembly OPTIONS... FASTQ_R1 FASTQ_R2

example
harpy metassembly --threads 20 -u prokaryote -k 13,51,75,83 FASTQ_R1 FASTQ_R2

# Running Options

In addition to the common runtime options , the assembly module is configured using the command-line arguments below. Since the assembly process consists of several distinct phases, the descriptions are provided with an extra badge to reflect which part of the assembly process they correspond to.

argument	short name	default	description
`FASTQ_R1`			required FASTQ file of forward reads
`FASTQ_R2`			required FASTQ file of reverse reads
`--extra-params`	`-x`		spades assembly Additional spades parameters, in quotes
`--kmer-length`	`-k`	`auto`	spades assembly Kmer lengths to use for initial spades assembly. They must be odd and <128, separated by commas, and without spaces. (e.g. `13,23,51`)
`--max-memory`	`-r`	`10000`	spades assembly Maximum memory for spades to use, given in megabytes
`--arcs-extra`	`-y`		arcs scaffold Additional ARCS parameters, in quotes and `option=arg` format
`--contig-length`	`-c`	`500`	arcs scaffold Minimum contig length
`--links`	`-n`	`5`	arcs scaffold Minimum number of links to compute scaffold
`--min-aligned`	`-a`	`5`	arcs scaffold Minimum aligned read pairs per barcode
`--min-quality`	`-q`	`0`	arcs scaffold Minimum mapping quality
`--mismatch`	`-m`	`5`	arcs scaffold Maximum number of mismatches
`--molecule-distance`	`-d`	`50000`	arcs scaffold Distance cutoff to split molecules (bp)
`--molecule-length`	`-l`	`2000`	arcs scaffold Minimum molecule length (bp)
`--seq-identity`	`-i`	`98`	arcs scaffold Minimum sequence identity
`--span`	`-s`	`20`	arcs scaffold Minimum number of spanning molecules to be considered assembled
`--organism-type`	`-u`	`eukaryote`	report Organism type for assembly report: `eukaryote`,`prokaryote`, or `fungus`

# Deconvolved Inputs

For linked-read assemblies, the barcodes need to be deconvolved in the sequence data, meaning that barcodes that are shared by reads that originate from different molecules need to have unique barcode IDs. Deconvolution often takes the form of adding a hyphenated integer to the end of a barcode so that software can recognize that they are different from each other. For example: two sequences from different molecules sharing the [linked read] barcode A03C45B11D91 would have one of them recoded as A03C45B11D91-1. Software like QuickDeconvolution, which is used by deconvolve will parse your fastq input files and perform this deconvolution.

# Assembly Workflow

Initial assembly is performed with cloudspades, followed by tigmint, arcs, links to scaffold the contig-level assembly.

graph LR
    subgraph Inputs
        F1([fastq read 1]):::clean
        F2([fastq read 2]):::clean
    end
    subgraph init[Initial Assembly]
        A([cloudspades]):::clean
    end
    Inputs--->A
    subgraph Scaffolding
        A ---> B([tigmint]):::clean
        B-->C([arcs]):::clean
        C-->D([links]):::clean
    end
    style Inputs fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
    style init fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
    style Scaffolding fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
    classDef clean fill:#f5f6f9,stroke:#b7c9ef,stroke-width:2px

The default output directory is Assembly with the folder structure below. Using --skip-reports will skip the QUAST/BUSCO analysis as well. The file structure below isn't exhaustive and serves to highlight the general structure and most important outputs.

Metassembly/
├── busco
│   ├── short_summary.*.txt
│   └── run_*_odb10
├── quast
│   ├── report.*
│   └── predicted_genes
├── reports
│   └── assembly.metrics.html
├── scaffold
├── spades
│   └── contigs.fasta
└── scaffolds.fasta

item	description
`busco/`	directory with results from the BUSCO analysis
`busco/short_summary.*.txt`	text file summarizing BUSCO analysis
`busco/run_*_odb10`	directory with results from the BUSCO analysis for the specific organism type
`scaffold/`	directory with the tigmint/arcs/links output
`scaffolds.fasta`	the resulting scaffolded assembly
`spades/`	directory with the SPADES output
`spades/contigs.fasta`	the resulting primary assembly
`quast/`	directory with results from the QUAST analysis
`quast/report*`	resulting QUAST report in various formats
`quast/predicted_genes/`	GLIMMER gene-finding output
`reports/assembly.metrics.html`	aggregate and generalization of QUAST and BUSCO results

By default, Harpy runs spades with these parameters (excluding inputs and outputs):

spades.py -t threads -m mem -k k --gemcode1-1 FQ_R1 --gemcode1-2 FQ_R2

See the SPADES documentation for a list of all available command line options.

These are the summary reports Harpy generates for this workflow. You may right-click the image and open it in a new tab if you wish to see the example in better detail.

Aggregated Report

QUAST Report

Aggregates QUAST and BUSCO analyses.

This is the report produced by QUAST