Modes

The configuration key mode selects the front end, the steps that turn the input into an assembly. The launch command does not change with the mode. This page sets the four modes side by side and compares their steps, the contamination and mitochondrion screen as each mode runs it, and the files each mode writes. Read it when you choose a mode, or to look up what a mode does.

From the screen on, every mode runs the same steps, and only polishing differs between the read-based modes. Every mode produces 02.assembly/{sample}/contigs_final.fasta. This file is the nuclear genome alone when the screen runs with the default parameters.mitochondria.mode: separate_confirmed. Otherwise nothing is split off, and any mitochondrial contig stays in it.

From that file on, all four modes run QUAST, BUSCO, ITS extraction and classification, repeat masking, gene prediction, functional annotation and the MultiQC report. Two of these steps take extra inputs in some modes. In hybrid mode QUAST also reports the Illumina guide assembly, and MultiQC adds the read-QC and mapping tables of the modes that have reads.

Terms used on this page

front end	the steps that differ by mode, chosen by mode: read processing, assembly and contig filter before the shared 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
contig selector	rule select_contigs, the last step of the screen: it keeps or removes each contig by genus, as parameters.decontamination says; the default, off, keeps every contig
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
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
assembler evidence	what hifiasm or Flye report for each contig, above all read depth and circularity, written to assembly_evidence.tsv for the mitochondrion screen; longread and hybrid only

The four modes side by side

Three modes build an assembly from reads, and contigs mode starts from one. The table compares the four modes step by step, and each column heading links to the page of its mode.

	illumina	longread	hybrid	contigs
Status	run on one public genome (Validation)	run on ONT reads of one public genome and PacBio HiFi reads of another, with hifiasm and with Flye	run on ONT and Illumina reads of one public genome and PacBio HiFi and short reads of another, with hifiasm; Flye in dry runs only	run on the assembly of one public genome (Validation)
Input	Illumina paired-end reads	ONT or PacBio HiFi reads, technology set per sample	Illumina pairs plus ONT or PacBio HiFi reads of the same isolate	an assembled genome (FASTA)
Input folder and file names	input.illumina_dir: {sample}_R1.<ext>, {sample}_R2.<ext>	input.longread_dir: {sample}_ont.<ext> or {sample}_pacbio.<ext>	both folders (may be the same one) and both sets of names	input.contigs_dir: {sample}.fasta, .fa or .fna
Other required keys	directories.blast_db, links.phix_link	directories.blast_db	directories.blast_db, links.phix_link	directories.blast_db, only with the screen
Mode settings	parameters.spades_kmers	parameters.longread	parameters.hybrid, parameters.spades_kmers	parameters.contigs
Read processing	PhiX removal (bowtie2), fastp	ONT adapters removed (fastplong); Filtlong; NanoPlot before and after	PhiX removal, fastp; ONT adapters removed (fastplong); Filtlong (ONT reads scored against the Illumina pairs only if you switch it on); NanoPlot	none
Assembly	SPAdes --isolate	hifiasm (default) or Flye	SPAdes guide assembly, then hifiasm or Flye	none
Contig filter	at least 500 bp and 2x coverage	none	guide assembly only: at least 500 bp and 2x	SPAdes headers: at least 500 bp and 2x; other headers: none
Polishing	none	Medaka, ONT samples	Medaka (ONT samples), then Polypolish with the Illumina pairs	none
Mapping QC	Qualimap	Qualimap	Qualimap, guide and long-read assembly	none
contigs_all.fasta is	the screened contigs	the Medaka consensus (ONT) or the screened assembly (PacBio HiFi, or Medaka off)	the Polypolish output	the screened FASTA; written only with the screen
Not run	long-read steps	PhiX removal, fastp, SPAdes, Polypolish; adapter trimming and Medaka for PacBio HiFi	adapter trimming, Medaka and Filtlong guidance for PacBio HiFi	read processing, assembly, polishing, mapping QC; the screen unless parameters.contigs.screen: true

contigs mode skips every read and assembly step and runs the screen only with parameters.contigs.screen: true. hybrid mode combines the other two read-based modes. Its Illumina steps build a guide assembly and select reads, its long-read steps build the genome that is delivered, and Polypolish corrects that genome with the Illumina pairs.

In longread and hybrid mode the read technology is taken from each file name, so one batch can hold ONT and PacBio HiFi samples. The flags that differ between the two technologies are listed on Long reads.

The contamination and mitochondrion screen

The screen checks every contig of the draft assembly. BLAST compares each contig with NCBI core_nt, and BlobTools gives it a genus. The mitochondrion screen looks for the mitochondrial genome among the contigs, and the contig selector keeps or removes each contig and records the decision. With the default parameters.mitochondria.mode: separate_confirmed, the contig selector keeps a confirmed mitochondrial contig whatever the genus filter says. Polishing comes after the screen, on the contigs that were kept.

The screen is the same in every mode. Three things change with the mode: the draft it examines, the reads behind the BlobTools coverage and the evidence for the mitochondrion.

	illumina	longread	hybrid	contigs with the screen
Draft screened	contigs_filt.fasta	assembly.fasta	the guide's contigs_filt.fasta, then assembly.fasta	contigs_filt.fasta
Reads behind the BlobTools coverage	trimmed Illumina pairs, bowtie2	filtered long reads, minimap2	guide: Illumina pairs, bowtie2; long-read assembly: filtered long reads, minimap2	none; the contigs are mapped to themselves and the coverage carries no information
Evidence for the mitochondrion	BLAST	BLAST plus the assembler's read depth and circularity	guide: BLAST; long-read assembly: BLAST plus assembler evidence	BLAST

A long-read assembler adds read depth and circularity to BLAST when the mitochondrion is judged. hybrid mode runs the screen twice, on the guide as in illumina and on the long-read assembly as in longread. In contigs mode there are no reads, so the BlobTools coverage carries no information and BLAST alone decides.

With the default settings, the screen removes no contig

The screen is on in every read-based mode and off in contigs mode unless parameters.contigs.screen: true. With the default parameters.decontamination.mode: off, it removes no contig but records a decision for each one and splits off the confirmed mitochondrion. See Decontamination and Mitochondria.

What 01.reads and 02.assembly contain

The table shows which folders and files each mode writes to 01.reads and 02.assembly. contigs_final.fasta, QUAST, BUSCO, the telomere files, the adapter screen and the contig summary are there in every mode. The other rows depend on each mode's reads, assembler, screen and polisher. Rows without a stage prefix are under 02.assembly/{sample}/, and 01.reads/ is not created in contigs mode.

Path	illumina	longread	hybrid	contigs
01.reads/{sample}/illumina/: fastp reports (kept), PhiX-free and trimmed reads (temporary)	yes		yes
01.reads/{sample}/long/: {sample}_filtered.fastq, NanoPlot raw_qc/ and filtered_qc/		yes	yes
01.reads/{sample}/long/adapters/: the adapter scan, fastplong's report and the check of the trimmed reads		ONT samples, unless ont_adapter: off	ONT samples, unless ont_adapter: off
01.reads/{sample}/selected/: Illumina pairs that map to the screened guide assembly			yes
02.assembly/{sample}/spades/	yes
02.assembly/{sample}/guide/: the Illumina guide assembly, its screen and Qualimap report			yes
hifiasm/ or flye/, assembly.fasta, assembly_evidence.tsv, assembly_provenance.tsv		yes	yes
contigs_filt.fasta	yes			with the screen
{sample}_medaka_model.txt, medaka/consensus.fasta		ONT samples with Medaka	ONT samples with Medaka
polypolish/polished.fasta			yes
contaminants/, mitochondria/	yes	yes	yes	with the screen
contigs_all.fasta, contigs_mitochondrial.fasta	yes	yes	yes	with the screen
contigs_final.fasta	yes	yes	yes	yes
eval/quast/, eval/busco/	yes	yes	yes	yes
eval/telomere_candidates.tsv, telomere_unit.txt, telomere_ends.tsv: the telomere repeat unit of the assembly and the arrays at the contig ends	yes	yes	yes	yes
eval/contig_summary.tsv, _mqc.tsv: what each contig of the draft is	yes	yes	yes	yes
eval/adapter_screen.tsv, adapter_screen_mqc.tsv: adapter and barcode sequence left in the draft	yes	yes	yes	yes
eval/qualimap/	yes	yes	yes

Every file is described on Output files.

Choosing a mode explains which mode fits a dataset.