Computational Tools
Command line workflows and tools for advanced users
There is so much to discover with HiFi reads. Beyond SMRT Link, which offers a graphic interface to execute fully-developed analysis workflows, PacBio research is constantly innovating with new tools that uncover more from HiFi sequencing.
This page highlights some of the popular tools and workflows for HiFi applications. To explore the complete list of tools developed by PacBio visit the PacBio GitHub page or access through Bioconda. Note that these command line tools are considered in-development tools, can be updated frequently, and are not supported as part of the PacBio software product suite.
Whole genome sequencing tools
These advanced variant callers and workflows provide a more comprehensive view of the full genome of an organism and exceptional accuracy for variant calling and complete genome assemblies.
RNA sequencing tools
Achieve isoform characterization of PacBio full-length RNA sequencing for bulk and single-cell transcriptome data. Characterize alternative splicing events, and discover new isoforms and fusion transcripts.
Microbiome sequencing tools
See microbiomes in high resolution with species or strain-level identification, complete genes and operons, and reference quality metagenome-assembled genomes.
Whole genome sequencing tools
These advanced variant callers and workflows provide a comprehensive view of the full genome of an organism and exceptional accuracy for variant calling and complete genome assemblies.
Variant calling
PacBio WGS Variant Pipeline
The recently developed PacBio WGS Variant Pipeline for human genome consolidates 11 robust secondary analysis tools into a best practice workflow for alignment, variant calling, joint calling and optional annotation.
This workflow is also accessible through our partner bioinformatics platforms:
Paraphase
Paraphase is a computational tool for variant calling in homologous genes, for either WGS or targeted sequencing. Paraphase was introduced for gene profiling of spinal muscular atrophy (SMA), for which it successfully identified full-length SMN1 and SMN2 haplotypes.
This tool is also accessible through our partner platform DNAnexus. This tool is also applicable for targeted sequencing.
Explore the SMA Case study and publication.1
TRGT
TRGT is a computational tool for targeted genotyping of tandem repeat variation from HiFi sequencing data. TRGT profiles sequence length and composition, mosaicism, and CpG methylation of each repeat. TRGT is accompanied by a visualization tool, TRVZ, and a database of allele sequences and methylation levels for nearly 1 million tandem repeats.
Explore the TRGT flier and publication.2
HiFiCNV
HiFiCNV is a copy number variant caller optimized for PacBio HiFi reads. This tool also provides utility for depth visualization.
pbsv
pbsv is a suite of tools to call and analyze structural variants in diploid genomes from HiFi reads. This tool is applicable for both WGS and targeted sequencing. Selected versions of pbSV are also available in the Structural Variant Calling analysis workflow of SMRT Link.
HiPhase
HiPhase is a tool that phases both small variants and structural variants made from HiFi sequencing data. HiPhase benefits from highly accurate long reads that allow phase blocks to span breaks from reference gaps and homozygous deletions.
Explore the HiPhase publication.
de novo assembly
As more researchers embrace the benefits of PacBio long-read sequencing technology for de novo genome assembly, an expanding community developed tools has taken shape.
Hifiasm
Developed by researchers at Dana-Farber Cancer Institute and Harvard Medical School, hifiasm is fast, easy to use and delivers high-quality telomere-to-telomere assemblies. It can by run on HiFi reads alone or combined with Hi-C reads or short-read parental data to enhance haplotype-resolved assemblies.
Explore the hifiasm publication.
Verkko
Developed by researchers at the National Human Genome Research Institute, verkko is an improved and automated version of the de novo assembly strategy used to produce a gapless human genome by the Telomere-to-Telomere consortium. Verkko uses a graph-based pipeline for assembling complete, diploid genomes.
Explore the verkko publication.
Whole genome datasets
| Application | Dataset | Download literature | Technology | Sequencing system |
|---|---|---|---|---|
| Variant detection, assembly, epigenetics | Homo sapiens — GIAB trio HG002-4 | N/A | HiFi long read | Revio system |
| Tumor/normal | COLO829 melanoma | N/A | HiFi long read | Revio system |
| Tumor/normal | HCC1395 | N/A | HiFi long read | Revio system |
| Whole genome sequencing | Various plant & animals – mouse, ladybug, oak, mistletoe, and maize | Plant + animal biology | HiFi long read | Revio system |
| Whole genome sequencing | Homo sapiens — HG002 | N/A | SBB short read | Onso system |
| Assembly (ultra-low DNA input) | Phlebotomus papatasi, Homo sapiens, Drosophila melanogaster | Considerations for using ultra-low DNA input workflows for WGS | HiFi long read | Sequel II system |
| Assembly | Food safety & infectious microbes – 96 plex | Microbial WGS | HiFi long read | Sequel IIe system |
RNA sequencing tools
Achieve isoform characterization of PacBio full-length RNA sequencing for bulk and single-cell transcriptome data. Characterize alternative splicing events, and discover new isoforms and fusion transcripts. For more details and tutorials on Iso-Seq tools please visit https://isoseq.how/.
IsoSeq
IsoSeq contains multiple sub-tools for full-length read identification, isoform clustering, and collapsing redundant transcripts. IsoSeq can be used with and without a reference genome.
pigeon
Pigeon is a long-read transcript classification and filtering tool based on SQANTI3. Full-length transcripts from the isoseq tool output can be classified against a known annotation (e.g., Gencode) to identify novel genes and isoforms. Additional sub-tools can be used to generate single-cell gene and isoform count matrices and saturation curves.
pbfusion
pbfusion is a fusion gene detection tool designed for identifying fusion transcripts from Iso-Seq data.
RNA sequencing datasets
| Application | Dataset | Download literature | Technology | Sequencing system |
|---|---|---|---|---|
| MAS-Seq single-cell | Homo sapiens - PBMC 10x Chromium Single Cell 3' libraries | MAS-Seq for single-cell isoform sequencing | HiFi long read | Sequel II and Revio systems |
| Whole transcriptome | Homo sapiens– brain with Alzheimer’s disease | Bulk and single-cell isoform sequencing for human disease research | HiFi long read | Sequel II system |
| Whole transcriptome | Homo sapiens – universal human reference RNA (UHRR) | Bulk and single-cell isoform sequencing for human disease research | HiFi long read | Sequel II system |
Microbiome sequencing
See microbiomes in high resolution with species level identification, complete genes and operons, and reference quality metagenome-assembled genomes.
pb-metagenomics-tools
pb-metagenomics-tools is a collection of recommended tools and common practices workflows for metagenomics profiling and assembly using HiFi reads.
Explore a list of metagenomics analysis using HiFi data.
Pb-16S-nf
The Pb-16S-nf Nextflow pipeline is designed to process HiFi full-length 16S data into high quality amplicon sequence variants (ASVs) using QIIME 2 and DADA2. This pipeline provides a set of visualizations through the QIIME 2 framework for interactive plotting and generates an HTML report for the important statistics and top taxonomies.
Metagenomics datasets
| Application | Dataset | Download literature | Technology | Sequencing system |
|---|---|---|---|---|
| Metagenomic profiling and assembly | ZymoBIOMICS Fecal Reference with TruMatrix Technology (human) | Microbiome and metagenome sequencing with HiFi reads | HiFi long read | Revio system |
| Metagenomic profiling and assembly | ZymoBIOMICS Fecal Reference with TruMatrix Technology (human) | Microbiome and metagenome sequencing with HiFi reads | HiFi long read | Sequel IIe system |
| Metagenomic profiling and assembly | 20 strain mock microbial community – ATCC MSA-1003 – shotgun | Microbiome and metagenome sequencing with HiFi reads | HiFi long read | Sequel II system |
| Metagenomic profiling and assembly | Human gut microbiome pooled standards | Microbiome and metagenome sequencing with HiFi reads | HiFi long read | Sequel IIe system |
| Full-length 16S sequencing | 20 strain mock microbial community – ATCC MSA-1003 – 16S | Microbiome and metagenome sequencing with HiFi reads | HiFi long read | Sequel II system |
- Publication: Chen X, Harting J, Farrow E, et al. Comprehensive SMN1 and SMN2 profiling for spinal muscular atrophy analysis using long-read PacBio HiFi sequencing. The American Journal of Human Genetics. 2023;0(0). doi:10.1016/j.ajhg.2023.01.001
- Publication: Dolzhenko, E., et al. (2023). Resolving the unsolved: Comprehensive assessment of tandem repeats at scale. bioRxiv 2023.05.12.540470.