Find the information short reads miss

Short reads can struggle to characterize complex structural variants, resolve full-length RNA isoforms, and assess methylation in repetitive regions. This guide covers how PacBio HiFi long-read sequencing is used for each, with published examples, planning checklists, and links to relevant analysis tools.

Find and phase structural variants with HiFi sequencing

See how HiFi sequencing can help resolve structural variants in complex regions of the genome. Get a seven-step workflow for planning an SV study, along with published comparisons, analysis tools, and guidance for evaluating your existing short-read samples.

Analyze DNA methylation and genetic variation together

HiFi sequencing can capture CpG methylation directly from native DNA, alongside sequence variants and haplotypes. This guide offers a seven-step workflow, analysis tools, and published research, with guidance for comparing HiFi methylation results against your existing array or bisulfite data.

Already sequencing native DNA with HiFi? Use this resource to see how to analyze methylation alongside your genomic data.

Identify full-length isoforms in plant and animal transcriptomes

Explore alternative splicing, novel isoforms, and transcript diversity with full-length HiFi reads. This guide covers the Kinnex and Iso-Seq method workflow, sample planning, and analysis tools, with a published zebrafish development example.

Published comparison

Across four genomes, long reads detected approximately 25,000 structural variants per genome, compared with 13,000 from short reads.

Source: Savara J et al., GigaScience (2026). Study-specific findings.

Published comparison

In a published comparison, 696,375 CpGs were reported only with HiFi, with approximately half located in centromeres.

Source: Ivashchenko V et al., Genome Medicine (2026). Study-specific findings.

Zebrafish development study

Full-length RNA sequencing identified 33,018 novel isoforms and 2,113 previously unannotated genes across 21 developmental time points.

Source: Bo J et al., BMC Biology (2025). Study-specific findings.

Cover of the guide: Move beyond short reads

Inside the guide

Three applications and a plan for your first study

Inside the guide

Explore structural variants, full-length isoforms, and DNA methylation, with a seven-step checklist for each application.

  • Structural variantsStructural variants

    Identify insertions, deletions, and complex rearrangements, including those in regions that are difficult to map with short reads.Published comparisons and tools for detecting, phasing, and genotyping complex SVs.

  • Full-length isoformsFull-length isoforms

    Identify known and novel isoforms and alternative splicing with the Iso-Seq method, without reconstructing transcripts from short fragments.Kinnex and Iso-Seq method workflows for exploring alternative splicing and novel transcripts.

  • Methylation from native DNAMethylation from native DNA

    Detect CpG methylation directly from native DNA, including in difficult-to-map regions, and examine methylation alongside sequence variants and haplotypes.Research and analysis tools for examining CpG methylation by region and haplotype.

  • Plan a HiFi studyPlanning your study

    Review sample requirements, coverage, library preparation, and analysis considerations for the application you’re evaluating.Seven-step checklists covering samples, library preparation, sequencing, and analysis for each application.

Is HiFi right for your study?

Find the application most relevant to your research question, then review the published evidence and planning considerations for that workflow.

See what HiFi could add to your existing data

Already have short-read data? Use the guide to plan a HiFi comparison on a few familiar samples, with a consistent approach to SV calling, phasing, and annotation.

Take a closer look at your methylation data

Have array or bisulfite data already? Use the guide to plan a region-by-region comparison with HiFi, including how to examine methylation alongside haplotypes and sequence variants.

Explore what full-length reads could reveal in your samples

Have samples from an existing RNA-seq study? Use the guide to plan full-length isoform analysis and compare what you identify with your current transcript annotation.

Get the guide

Illustration: short reads stop at a repeat region, while one HiFi read spans the repeat and the insertion inside itIllustration: short reads stop at a repeat region, while one HiFi read spans the repeat and the insertion inside it
Illustrative, not data. In repetitive regions short reads cannot map reliably. One HiFi read spans the repeat and the insertion inside it.
Cover of the guide: Move beyond short reads

Get the guide

Get the HiFi sequencing guide

It includes information about SVs, full-length isoforms, and methylation.

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