Long-read Sequencing

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Long-Read Platform Comparison

PacBio HiFi vs Oxford Nanopore

🎯PacBio HiFi15-25 kb reads | >99.9% accuracy | $30-50/Gb | Methylation (5mC) | Best for clinical-grade SV calling

⚡Nanopore R1010 kb-1 Mb reads | 98-99% accuracy | $5-10/Gb | Real-time | Direct methylation (5mC, 5hmC, 6mA)

🏆Key AdvantagesPhasing | STR genotyping | Full-length transcripts | Centromere/telomere assembly | Complex SV resolution

PacBio HiFi vs Nanopore

PacBio HiFi (CCS): 15–25 kb reads; >99.9% single-molecule accuracy; SMRT cells on Revio/Sequel IIe
PacBio raw reads (CLR): 10–100 kb; ~85–90% accuracy; used for de novo assembly
Oxford Nanopore (R10.4.1): 10 kb–Mb reads; 98–99% accuracy with latest Dorado/POD5
Nanopore direct DNA: preserves methylation (5mC, 5hmC, 6mA) - no bisulfite conversion
Nanopore adaptive sampling: selectively sequence or reject reads in real-time
Cost comparison: PacBio HiFi ~$30–50/Gb; Nanopore PromethION ~$5–10/Gb

Long-read Alignment

code

# Minimap2 - universal long-read aligner
# For PacBio HiFi
minimap2 -ax map-hifi -t 16 \
  hg38.fa sample.fastq.gz \
  | samtools sort -@ 8 -o hifi.bam
samtools index hifi.bam

# For Nanopore (R10)
minimap2 -ax map-ont -t 16 \
  hg38.fa sample.fastq.gz \
  | samtools sort -@ 8 -o ont.bam

# PBMM2 - PacBio native aligner (Minimap2-based)
pbmm2 align hg38.fa sample.bam aligned.bam \
  --preset HiFi --sort -j 16 \
  --sample patient001

Unique Long-read Applications

Haplotype phasing: heterozygous variants separated into maternal/paternal haplotypes (WhatsHap, HiPhase)
STR genotyping: tandem repeat expansion calls with base-pair precision (TRGT, STRique)
Methylation: direct detection of 5mC/5hmC with Dorado (no bisulfite conversion needed)
Full-length transcripts: Iso-seq (PacBio) or cDNA-PCR (Nanopore) capture complete splice variants
Centromere/telomere assembly: resolves regions invisible to short reads
De novo assembly: hifiasm + HiC scaffolding achieves T2T quality genomes

Nanopore Basecalling

code

# Dorado (Oxford Nanopore Technologies - current standard)
dorado basecaller \
  [email protected] \
  /path/to/pod5_files/ \
  --reference hg38.mmi \
  --modified-bases 5mCG_5hmCG \
  > calls.bam

# Convert to FASTQ
samtools fastq calls.bam > calls.fastq.gz

# Quality filtering
filtlong --min_length 1000 --min_mean_q 90 \
  calls.fastq.gz > filtered.fastq.gz

Structural Variant Analysis Python for Genomics