Scientific posters

Targeted long-read sequencing enables efficient characterization of tandem repeats, structural variants, and copy number variants that currently require non-NGS assays like rpPCR, MLPA, long range PCR, and Sanger sequencing. Here we demonstrate that our PCR-free Cas-9 target enrichment panels can resolve these variants in clinically- relevant regions of the genome.

Selecting the appropriate genomic coverage for reference-based variant detection requires balancing sequencing cost with accuracy. While 30x coverage is the standard for comprehensive variant detection with short-read technologies, it remains unclear whether this benchmark applies to long-read sequencing, which offers improved coverage uniformity, access to challenging genomic regions, and haplotype resolution. To address this, we analyzed a 40x HiFi dataset of HG002 generated on a single Revio SMRT Cell using PacBio’s new SPRQ chemistry. We downsampled the data to simulate varying coverage levels and assessed small and structural variant calling accuracy against Genome in a Bottle (GIAB) benchmarks.

Saliva stabilized in Oragene devices and extracted with Nanobind kits is a convenient sample type for HiFi long-read sequencing and broadly applicable to large-scale genomic studies. WGS from saliva samples show optimal HiFi performance with 20-fold to 40-fold coverage sufficient for comprehensive variant detection and methylation analysis. PureTarget from saliva samples enables characterization of complex genes for carrier screening using an accessible sample type.

Mitorsaw is an analysis tool for HiFi sequencing data that: Generates full-length mitochondrial haplotype sequences, reports small and structural variants with allele fractions, recovers variants near the edge of the reference, removes false positives caused by NUMTs and creates custom visualizations for manual inspection

The latest release of the Platinum Pedigree truth set spans the full spectrum of variation (SNVs, indels, tandem repeats, and structural variants) and is further improved by the integration of genome assemblies. As the field shifts toward sequence-resolved haplotypes, new methods for merging and scoring are needed to capture complex variation in difficult regions. Aardvark fills this gap with its basepair-based accuracy measure.

The D4Z4 repeat is a variable number tandem repeat (VNTR) that contains some of the most difficult-to-resolve medically relevant alleles in the human genome. D4Z4 has a repeat unit of 3.3 kb (encoding the DUX4 gene) that is repeated between 1-100 times per allele. Facioscapulohumeral muscular dystrophy (FSHD) is caused by ectopic expression of DUX4, mediated by contraction of D4Z4 to less than 11 copies (FSHD1, 95% of FSHD cases) or hypomethylation of D4Z4 induced by mutations in the epigenetic machinery such as SMCHD1 (FSHD2, 5% of FSHD cases). D4Z4 is present on both chromosome 4 and chromosome 10, while DUX4 can only be expressed from the permissive A haplotype that usually occurs on chromosome 4. The D4Z4 repeat size is conventionally measured by Southern blot analysis, which is low-throughput and can be confounded by genetic polymorphism. Due to the large repeat size, high polymorphism and homology issues, D4Z4 remains difficult to resolve by sequencing technologies and population analysis is lacking. Here we present a computational tool, Kivvi, to resolve D4Z4 using PacBio HiFi whole-genome sequence data.

Paraviewer enhances interpretation of Paraphase results. Many medically relevant genes fall in regions where high sequence homology (paralogs/pseudogenes) limits reference- based variant calling. Paraphase1 phases haplotypes for genes in the same family, determines copy numbers, and reports phased variant calls. Paraphase reports variant calls in 160 segmental duplications, including 11 medically relevant regions.

Metagenome assembly of human oral microbiomes can be difficult using short reads due to strain-level diversity and high levels of host contamination (30% of reads for tongue scrape and 80% for saliva4). Here, we demonstrate that HiFi data obtained from the high-throughput PacBio Revio system is low in host contamination and sufficient to assemble high-quality MAGs from the oral microbiome.

Saliva samples collected with OrageneTM devices and DNA extracted using Nanobind kits are a good alternative to blood for HiFi sequencing. High-throughput workflow from extraction using Nanobind HT kit through HiFi sequencing on the Revio system is available for saliva and blood samples.

Sawfish is a general-purpose structural variant (SV) caller for HiFi sequencing data. It has already been shown to provide best-in- class SV accuracy in both single-sample and joint-genotyping contexts1 . Sawfish2 adds depth-based CNV calling as a joint operation integrated with sawfish’s existing breakend-based SV calling methods.

Here, we combine IDT hybridization capture with PacBio full-length RNA sequencing for deep isoform characterization. We show that the new IDT xGen Hyb and Wash Kit v3 is compatible with the PacBio Kinnex full-length RNA kit and can achieve an on-target rate of 85%, increasing detection of low abundance isoforms by several thousand-fold.

Studies of structural variants (SVs) in large cohorts remain challenging due to high data volumes and imprecise breakpoints in low sequence complexity regions, such as tandem repeats. We developed SVX, a fast, memory-efficient SV merging tool implemented in Rust. SVX is in early development. It currently only works with Sawfish.

Despite advances in DNA sequencing, the causal mutations of many human diseases remain challenging to characterize with standard methods. The adoption of long-read sequencing can improve and simplify sequencing and analysis of these complex regions. The ability to detect methylation enables simultaneous generation of sequence- level and epigenetic data. We present new panel content for the PureTarget assay, which uses the CRISPR/Cas9 system to generate targeted sequencing libraries of native DNA, sequenced with long and accurate HiFi sequencing.

Here we apply PacBio HiFi to perform whole-genome sequencing of the newly described HG008 matched tumor- normal pair from the Genome in a Bottle (GIAB) consortium. 1 This reference sample includes an adherent, epithelial-like pancreatic adenocarcinoma (PDAC) cell line as the tumor material, with the matched normal obtained from adjacent duodenal and pancreatic tissue. We perform whole-genome sequencing of the tumor cell line and matched pancreatic normal tissue with PacBio HiFi, resulting in a more robust and comprehensive picture of somatic variation in this reference sample and contributing to the development of this novel benchmark.

The characterization of somatic variation, especially in complex genomic regions, is crucial for understanding the molecular drivers of cancer progression. Accurate PacBio long-read sequencing (HiFi) enables detection of all variant classes, from simple SNVs and INDELs up to complex structural variation, tandem repeats, and changes in epigenetic signatures. Complex and repetitive regions, while fully sequenced by HiFi reads, remain bioinformatically challenging, requiring tailored solutions. Here, we describe new tools to genotype understudied repetitive regions in cancer genomes, a task that has historically posed significant challenges for short-read sequencing.