Assembles haplotype-resolved diploid and telomere-to-telomere (T2T) genomes from PacBio HiFi reads with hifiasm (HiFi-only, Hi-C, or trio phasing) and verkko (HiFi + ultralong ONT for T2T), extracting contigs from GFA and routing phasing QC to k-mer/trio metrics. Covers why a primary assembly is a haplotype mosaic that exists in no cell, partial-vs-full phasing (the .bp. vs .dip. filename convention), the purge-default trap on inbred samples, the --hom-cov coverage-estimate alarm, and verkko-vs-hifiasm for T2T. Use when assembling a diploid eukaryote from HiFi, phasing haplotypes with parents (trio) or Hi-C, deciding whether to chase T2T, or diagnosing switch errors invisible to N50/BUSCO/QV.
Assembles haplotype-resolved diploid and telomere-to-telomere (T2T) genomes from PacBio HiFi reads with hifiasm (HiFi-only, Hi-C, or trio phasing) and verkko (HiFi + ultralong ONT for T2T), extracting contigs from GFA and routing phasing QC to k-mer/trio metrics. Covers why a primary assembly is a haplotype mosaic that exists in no cell, partial-vs-full phasing (the .bp. vs .dip. filename convention), the purge-default trap on inbred samples, the --hom-cov coverage-estimate alarm, and verkko-vs-hifiasm for T2T. Use when assembling a diploid eukaryote from HiFi, phasing haplotypes with parents (trio) or Hi-C, deciding whether to chase T2T, or diagnosing switch errors invisible to N50/BUSCO/QV.
Before using code patterns, verify installed versions match. If versions differ:
CLI: <tool> --version then <tool> --help to confirm flags
hifiasm output filenames AND the default purge level are version-dependent: confirm the .bp.*/.dip.* prefixes and -l defaults against hifiasm --help and the man page for the installed build before scripting around them. The k-mer-size convention also matters more than the binary version: hifiasm/yak trio uses k=31; verkko/Merqury hap-mers commonly use k=30 - mixing them silently produces garbage hap-mer matching. If a command errors, introspect the installed tool and adapt rather than retrying.
HiFi Assembly
"Assemble a diploid genome from HiFi reads" -> Build two cleanly phased haplotypes (not one mosaic primary) from accurate long reads, choosing the phasing mechanism from the sample type and available data, and validate phasing with k-mer QC the headline metrics cannot see.
The Single Most Important Modern Insight -- A Primary Assembly Is a Mosaic Chimera, Not a Haplotype
A "primary assembly" is not a genome that exists in any cell. At every heterozygous block the assembler picks one allele, and which one it picks switches arbitrarily from block to block - so the primary is a stitched chimera: maternal here, paternal there, matching no gamete, no parent, no individual. It is a fine haploid representation for "roughly where is gene X" and a terrible substrate for anything allele-aware (phased variant calling, allele-specific expression, HLA/KIR typing, compound-heterozygote analysis). HiFi's combination of length AND ~Q30 accuracy made phased diploid assembly routine, so the field's deliverable shifted from one collapsed reference to two phased haplotypes (and onward to the pangenome: each HPRC node is a per-sample phased diploid assembly, Liao 2023 Nature 617:312).
The killer corollary: the thing that is wrong with a mosaic - that it is a haplotype mix - is exactly the thing none of the metrics people check can see. A switch error does not break a contig (N50 unchanged), delete a gene (BUSCO unchanged), or introduce a wrong base (QV unchanged - both alleles are real sequence, just assigned to the wrong haplotype). Switch errors and haplotype mosaicism are structurally invisible to N50, BUSCO, and even base-level QV. Only k-mer/trio QC - Merqury hap-mer blob plots and switch/hamming error against parental k-mers - can see them. "Reported N50 and BUSCO but no switch/hamming or hap-mer plot" means the phasing was never validated. Subtle trap: the HiFi-only .bp.hap1/hap2 are partially phased (locally phased, switch errors between blocks) - "hap1" in a filename does NOT certify global phasing; the phasing data supplied (trio or Hi-C) does.
Key flags:-l purge level (0 none, 1 light, 2/3 aggressive; default 3 in HiFi-only, 0 in trio); --h1/--h2 Hi-C R1/R2; -1/-2 paternal/maternal yak DBs; --ul ultralong ONT; --hom-cov INT force the homozygous-coverage peak; --n-hap INT ploidy (default 2); --primary emit primary + alternate (a_ctg) instead of dual hap1/hap2.
Output Filenames: the .bp. vs .dip. Convention (read the prefix, it encodes the mode)
The prefix is not cosmetic - it tells how the assembly was phased:
.bp. ("balanced phasing") = HiFi-only OR Hi-C mode.prefix.bp.p_ctg.gfa (the mosaic primary), prefix.bp.hap1.p_ctg.gfa/prefix.bp.hap2.p_ctg.gfa (the two haplotypes - partially phased in HiFi-only, fully phased with Hi-C). Also prefix.bp.r_utg.gfa/p_utg.gfa (unitig graphs).
--primary mode:prefix.p_ctg.gfa (primary) + prefix.a_ctg.gfa (alternate). The alternate is incomplete by construction (only heterozygous loci produce alt contigs).
Reusable binariesprefix.ec.bin/ovlp.*.bin let a re-run with different -l/phasing skip error-correction.
People grep hap1.p_ctg and are confused when a trio run made dip.hap1.p_ctg and a Hi-C run made bp.hap1.p_ctg. Verify the actual emitted names against the installed version.
GFA Is Not FASTA (extract S lines before anything downstream)
hifiasm and verkko emit assembly graphs (GFA), and downstream tools want FASTA. Contig sequences live in GFA S (segment) lines:
The graph also carries bubbles/alternate paths the FASTA throws away - keep the GFA. Verkko works internally in homopolymer-compressed coordinates (its .gfa is HPC); its final assembly.fasta is in normal space.
verkko (T2T, when the goal genuinely needs it)
T2T is a project, not a flag. Reach for verkko only when T2T completeness is the actual goal AND the data exist (deep HiFi PLUS good ultralong ONT N50 PLUS trio or Hi-C). It is slower, heavier (hundreds of CPU-hours, high RAM, Snakemake-orchestrated), and more fragile than hifiasm. CHM13 - the first T2T human - was a hydatidiform mole precisely because a mole is effectively homozygous, decoupling repeat-resolution from phasing (Nurk 2022 Science 376:44).
--hifi accurate reads; --nano ultralong ONT (the spanning data, strongly recommended); --hap-kmers <hap1> <hap2> trio (argument ORDER sets which becomes haplotype1/haplotype2 - it is not inferred from biology) or --hic1/--hic2 for phasing. Outputs assembly.fasta, assembly.haplotype1.fasta, assembly.haplotype2.fasta, assembly.homopolymer-compressed.gfa. verkko-vs-hifiasm(--ul): verkko closes more chromosomes automatically at higher cost; hifiasm --ul brings much of the spanning benefit far cheaper but needs more manual finishing. Neither reaches T2T without ultralong reads.
Per-Method Failure Modes
Primary assembly used as if it were a haplotype
Trigger: running the primary .bp.p_ctg/p_ctg into an allele-aware analysis. Mechanism: the primary is a per-block haplotype mosaic. Symptom: garbage phased variant / ASE / HLA results; "best" metrics. Fix: use trio/Hi-C-phased hap1/hap2; never the primary for allele-aware work.
Partial phasing mistaken for full phasing
Trigger: treating HiFi-only .bp.hap1/hap2 as trio-grade haplotypes. Mechanism: no linkage data -> locally phased with inter-block switch errors. Symptom: phasing breaks at block boundaries; high switch rate vs trio. Fix: add Hi-C (--h1/--h2) or trio (-1/-2) for global phasing; validate with hap-mers.
Default purging on an inbred/homozygous sample
Trigger: running hifiasm at default -l3 on an inbred line, doubled-haploid, or mole. Mechanism: real segmental duplications/paralogs look like duplicate haplotigs in a homozygous genome and get purged. Symptom: assembly shrinks below true genome size; real duplications collapsed. Fix:-l0 (purging off) for low-heterozygosity samples.
Unbalanced hap1/hap2 size read as biology
Trigger: one haplotype much larger than the other. Mechanism: hifiasm mis-estimated the homozygous-coverage peak (bimodal coverage, odd ploidy, contamination), so it over-/under-purged. Symptom: size ratio far from 1. Fix: read the peak off the k-mer/coverage histogram (GenomeScope) and set --hom-cov.
Phasing reported without phasing QC
Trigger: N50 + BUSCO + QV reported, no switch/hamming or hap-mer blob plot. Mechanism: those metrics are structurally blind to mosaicism. Symptom: a "great" assembly that is a phasing disaster. Fix: Merqury hap-mer blob plot + switch/hamming vs trio/Hi-C hap-mers (-> assembly-qc). No hap-mers (no trio/Hi-C) = phasing unvalidated by construction.
verkko recommended reflexively / without ultralong reads
Trigger: reaching for verkko (or --ul) without ultralong ONT or without a T2T goal. Mechanism: UL N50 - not HiFi coverage - is the binding T2T constraint; 60x HiFi cannot span a 3 Mb satellite array. Symptom: months of compute, no T2T gain. Fix: confirm UL N50 (a meaningful fraction >100 kb) and a real T2T need first; else hifiasm-HiFi-only.
Quantitative Thresholds
Threshold
Source
Rationale
HiFi coverage >=13x per haplotype; ~30-40x diploid sweet spot
hifiasm FAQ + community
below ~13x/hap het bubbles fragment; past ~40x diminishing returns and worse false-dup
HiFi >40x is not free quality
community convention
extra coverage can confuse the --hom-cov estimate and inflate false duplications
Ultralong ONT N50: the longer the better, meaningful fraction >100 kb (T2T targets >100 kb-1 Mb)
T2T practice
UL value is entirely about spanning centromeres/satellites; short "long" reads add little
-l purge: default 3 (HiFi-only), 0 (trio), set 0 for inbred
hifiasm man page
aggressive purging cleans outbred het-dups but deletes real sequence in homozygous samples
k=31 (yak/hifiasm trio) vs k=30 (verkko/Merqury hap-mers)
tool conventions
mixing k silently corrupts hap-mer matching - match each tool
UL N50 is the binding constraint; add long UL or stop at hifiasm
Trio run produced dip.*, scripts expected bp.*
mode encodes the prefix
match filename prefix to phasing mode
References
Cheng H, Concepcion GT, Feng X, Zhang H, Li H. 2021. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm. Nat Methods 18:170-175.
Cheng H, Jarvis ED, Fedrigo O, et al. 2022. Haplotype-resolved assembly of diploid genomes without parental data. Nat Biotechnol 40:1332-1335.
Nurk S, Walenz BP, Rhie A, et al. 2020. HiCanu: accurate assembly of segmental duplications, satellites, and allelic variants from high-fidelity long reads. Genome Res 30:1291-1305.
Koren S, Rhie A, Walenz BP, et al. 2018. De novo assembly of haplotype-resolved genomes with trio binning. Nat Biotechnol 36:1174-1182.
Rautiainen M, Nurk S, Walenz BP, et al. 2023. Telomere-to-telomere assembly of diploid chromosomes with Verkko. Nat Biotechnol 41:1474-1482.
Antipov D, Rautiainen M, Nurk S, et al. 2025. Verkko2 integrates proximity-ligation data with long-read de Bruijn graphs. Genome Res 35:1583-1594.
Nurk S, Koren S, Rhie A, et al. 2022. The complete sequence of a human genome (T2T-CHM13). Science 376:44-53.
Liao WW, Asri M, Ebler J, et al. 2023. A draft human pangenome reference. Nature 617:312-324.
Rhie A, Walenz BP, Koren S, Phillippy AM. 2020. Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies. Genome Biol 21:245.
Related Skills
genome-profiling - Decide outbred/inbred/ploidy and the homozygous-coverage peak before setting purge level and --hom-cov
assembly-qc - Merqury hap-mer blob plots and switch/hamming are the only QC that sees phasing
assembly-polishing - HiFi is already accurate; deciding whether to polish at all
scaffolding - Hi-C used here for phasing; chromosome-scale scaffolding (YaHS/SALSA2) is the adjacent step
contamination-detection - Screen contigs for foreign sequence after assembly
long-read-sequencing/long-read-qc - HiFi read length/QV/contamination before assembly
