Rationale

This page explains why FunFlux is built as it is. One workflow takes four kinds of input, and only its front end depends on the mode. The page also explains why the mitochondrial genome is kept out of the nuclear genome. Read it to understand what the mode key changes and what it leaves the same.

Terms used on this page

front end	the steps that differ by mode, chosen by mode: read processing, assembly and contig filter before the screen, and polishing after it
the screen	the contamination and mitochondrion screen of the draft: BLAST against core_nt, BlobTools, the mitochondrion screen and the contig selector; in contigs mode only with parameters.contigs.screen: true
draft assembly (the draft)	the assembly the screen examines, before any contig is removed: contigs_filt.fasta in illumina mode and in contigs mode with the screen, assembly.fasta in longread and hybrid
guide assembly	in hybrid mode, the SPAdes assembly of the Illumina reads; it is screened, and only the pairs that map to it go on
coverage track	the reads aligned back to the draft, contaminants/{sample}_map.bam, from which BlobTools takes each contig's coverage and the contig summary its read depth; in contigs mode, the contigs aligned to themselves
genus filter	the setting parameters.decontamination.mode: include or exclude, under which the contig selector keeps or removes contigs by the genus BlobTools gave them
confirmed mitochondrial contig	a contig the mitochondrion screen classes confirmed_mitochondrial; with the default separate_confirmed, it is kept whatever its genus and moved to contigs_mitochondrial.fasta
nuclear genome	the delivered genome without the confirmed mitochondrial contigs, contigs_final.fasta; it holds every contig when nothing is split off (no screen, or parameters.mitochondria.mode off or report)

One workflow instead of four

FunFlux 2.0.0 joins four earlier workflows into one, and the mode key chooses which kind of input it starts from. Coming from v1 lists what changed for a v1 user.

What is shared and what depends on the mode

Only the front end differs between the modes:

Part	Steps
Front end: differs by mode	read QC, read filtering, assembly and the contig filter before the screen, and polishing after it; in hybrid also the Illumina guide assembly, its own screen, and the selection of the Illumina read pairs for Polypolish and, only with short_read_guidance: true (off by default), for Filtlong on ONT samples
Shared: the same in every mode	the contamination and mitochondrion screen, the split into contigs_final.fasta and contigs_mitochondrial.fasta, QUAST and BUSCO, the adapter screen, the contig summary, the ITS extraction and classification, repeat masking, gene prediction, functional annotation, the AMR screen and the MultiQC report

From the screen on, every mode runs the same code, and only polishing differs between the read-based modes. The stage folders, 02.assembly to 06.report, have the same names in every mode. Where a shared step depends on the data, it follows what the run has. The coverage track of the screen, for example, comes from the reads the run has, or from the contigs themselves in contigs mode, as described on Decontamination.

The long-read technology is not a mode of its own. It is a property of each sample, read from the file name (_ont or _pacbio), so one longread or hybrid batch can hold both. See One technology per sample.

Why the mitochondrion is kept out of the nuclear genome

In every mode the assembly stage ends with 02.assembly/{sample}/contigs_final.fasta, and QUAST, BUSCO, the ITS extraction and funannotate read that file. The mitochondrial genome has to stay out of these steps, because fungal mitochondria use a different genetic code. The mitochondria of most fungi use code 4, in which UGA codes for tryptophan instead of stop, and those of Saccharomyces cerevisiae and related yeasts use code 3. On a mitochondrial contig, the nuclear gene predictors would call false genes.

contigs_final.fasta is the nuclear genome alone when the screen runs with the default parameters.mitochondria.mode: separate_confirmed. Otherwise it holds every contig, a mitochondrion included.

The steps that set the mitochondrion apart run in this order:

1. The mitochondrion screen runs on the draft assembly.
2. With the default separate_confirmed, the confirmed mitochondrial contigs are protected from the genus filter, because BLAST can assign a fungal mitochondrion to another genus.
3. Polishing comes next, in the long-read modes only. In longread, Medaka polishes the screened assembly of ONT samples unless medaka_model: false switches it off, and PacBio HiFi samples are not polished. In hybrid, Polypolish runs for every sample, after Medaka for ONT samples with Medaka on.
4. Only after that are the contigs split into contigs_final.fasta and contigs_mitochondrial.fasta, so the mitochondrion is polished together with the rest wherever a sample is polished. contigs_all.fasta keeps the two together.

See Mitochondria for the mitochondrion screen.