# Create a Metagenome 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 mixed-sample data, you might be interested in a metagenome assembly, also known as a metassembly. Unlike a single-sample assembly, a metassembly assumes there are multiple genomes present in your sequences and will try to assemble the most contiguous sequences for multi-sample (or multi-species) data.

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 metassembly module is configured using these command-line arguments:

argument	short name	default	description
`FASTQ_R1`			required deconvolved FASTQ file of forward reads
`FASTQ_R2`			required deconvolved FASTQ file of reverse reads
`--bx-tag`	`-b`	`BX`	required Which sequence header tag encodes the linked-read barcode (`BX` for `BX:Z` or `BC` for `BC:Z`)
`--extra-params`	`-x`		Additional spades parameters, in quotes
`--ignore-bx`			Ignore linked-read info for initial spades assembly
`--kmer-length`	`-k`	`auto`	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`	Maximum memory for spades to use, given in megabytes
`--organism-type`	`-u`	`eukaryote`	Organism type for assembly report. Options: `eukaryote`,`prokaryote`,`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.

# Metassembly Workflow

Initial assembly is performed with spades or cloudspades depending on whether --ignore-bx was used. After the initial spades-based assembly, athena assembles the contigs into larger scaffolds.

graph LR
    subgraph Inputs
        F1([fastq forward]):::clean
        F2([fastq reverse]):::clean
        F1---|and|F2
    end
    subgraph init[Initial Assembly]
        AC([meta cloudspades]):::clean
        A([metaspades]):::clean
        A---|or|AC
    end
    subgraph athena[Secondary Assembly]
        B([athena]):::clean
    end
    Inputs ---> sort([sort by barcode]):::clean
    sort--->init--->B
    style Inputs fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
    style init fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
    style athena fill:#f0f0f0,stroke:#e8e8e8,stroke-width:2px
    classDef clean fill:#f5f6f9,stroke:#b7c9ef,stroke-width:2px

The default output directory is Metassembly with the folder structure below. If --ignore-bx is used, the initial spades assembly will be in */spades_assembly, otherwise it will be in */cloudspades_assembly. 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/
├── athena
│   ├── athena.asm.fa
│   └── athena.config
├── busco
│   ├── short_summary.*.txt
│   └── run_*_odb10
├── quast
│   ├── report.*
│   └── predicted_genes
├── reports
│   └── assembly.metrics.html
└── *spades_assembly
    └── contigs.fasta

item	description
`athena/athena.asm.fa`	final metagenome assemble
`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
`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
`*spades_assembly/`	directory with the SPADES output
`*spades_assembly/contigs.fasta`	the resulting primary assembly

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

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

# with --ignore-bx
## error correct reads
metaspades.py -t threads -m mem -k k -1 FQ_R1 -2 FQ_R2 --only-error-correction
## assemble corrected reads
metaspades.py -t threads -m mem -k k -1 FQ_R1C -2 FQ_R2C -s FQ_UNPAIREDC --only-assembler

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