What is Long Read Sequencing (PacBio)?

Long Read Sequencing with PacBio SMRT (Single Molecule, Real-Time) technology is a cutting-edge approach to sequencing DNA molecules in their full length. Unlike short-read technologies (like Illumina) that generate reads of 100–300 bp, PacBio long-read sequencing routinely produces reads of 10–25 kb, and in some cases, >50 kb.

This technology offers unparalleled advantages in genome assembly, structural variant detection, haplotype phasing, and resolving complex genomic regions.

Overview

• Platform: PacBio Sequel IIe / Sequel II / RS II

• Technology: SMRT (Single Molecule, Real-Time) Sequencing

• Read Lengths: Average 15–25 kb; Maximum >100 kb

• Key Strengths: Long reads, high accuracy (HiFi reads), structural variant detection, de novo assembly

How PacBio Long-Read Sequencing Works

1. High-Molecular-Weight DNA Extraction
   Requires high-quality, long DNA molecules (10 kb+ preferred).

2. SMRTbell Library Preparation
   DNA fragments are ligated with hairpin adapters at both ends, forming circular SMRTbell templates.

3. Real-Time Sequencing
   DNA polymerase incorporates nucleotides in real-time within Zero-Mode Waveguides (ZMWs) — a proprietary nanowell technology that tracks base incorporation.

4. Circular Consensus Sequencing (CCS)
   The polymerase reads the circular template multiple times to produce a HiFi (High-Fidelity) read — extremely accurate long reads with >99.9% accuracy.

5. Data Output & Analysis
   Output includes raw subreads, consensus HiFi reads, and detailed information on structural variations and base modifications.

Applications of PacBio Long-Read Sequencing

• De Novo Genome Assembly
  Produces complete, contiguous assemblies without gaps.

• Structural Variant Detection
  Detects large insertions, deletions, inversions, duplications, and translocations.

• Full-Length Transcript Sequencing (Iso-Seq)
  Captures complete mRNA isoforms without assembly.

• Haplotype Phasing
  Resolves maternal vs. paternal alleles in diploid genomes.

• Repeat Resolution
  Efficiently spans long repeat regions and complex genomic loci.

• Microbial and Metagenome Assembly
  Resolves entire bacterial chromosomes or plasmids with fewer contigs.

Key Features of PacBio Long Read Sequencing

Advantages of PacBio Long Read Sequencing

• Superior Genome Assembly
  Resolves complex genomic architectures and repetitive regions.

• High Accuracy with HiFi Reads
  Offers both long read length and Illumina-like base-level precision.

• Structural Variant Detection
  Ideal for finding large SVs that short reads miss.

• Minimal Bias
  No PCR amplification reduces bias across GC-rich or repetitive regions.

• Phasing and Methylation
  Simultaneously captures epigenetic marks and distinguishes haplotypes.

Limitations and Considerations

• High-Quality Input DNA Required
  Fragmented or degraded DNA reduces read length.

• Higher Cost Per Gb
  More expensive than short-read technologies, though more informative.

• Lower Read Count per Run
  Fewer total reads compared to high-throughput short-read platforms.

• Complex Library Preparation
  Requires precision and care, especially for ultra-long reads.

Bioinformatics Tools for PacBio Long Reads

• SMRT Link – PacBio’s official suite for data analysis (base calling, mapping, variant calling)

• Canu / HiCanu – De novo genome assembly tailored for long reads

• Flye – Fast, efficient long-read genome assembler

• hifiasm – Specialized assembler for HiFi reads

• pbsv – PacBio tool for structural variant calling

• DeepVariant – Google’s variant caller adapted for PacBio HiFi data

• Long Ranger / WhatsHap – Phasing and variant calling tools

Who Uses PacBio Long Read Sequencing?

• Genome Researchers – Building complete genome assemblies for humans, plants, animals, microbes

• Cancer Genomics Labs – Identifying structural variants, fusions, and complex mutations

• Evolutionary Biologists – Phasing genomes and analyzing genetic diversity

• Agrigenomics Teams – Sequencing crops and livestock genomes for breeding research

• Microbiologists – Assembling complete genomes from environmental samples

Comparison: PacBio vs Other Sequencing Platforms