Comprehensive SMN1 and SMN2 profiling for spinal muscular atrophy analysis using long-read PacBio HiFi sequencing

Chen, Xiao; Harting, John; Farrow, Emily; Thiffault, Isabelle; Kasperavičiūtė, Dalia; Hoischen, Alexander; Gilissen, Christian; Pastinen, Tomi; Eberle, Michael A.

doi:10.1016/j.ajhg.2023.01.001

Cited by 32 publications

(26 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our workflow which enables parallel analysis of multiple samples, implements several new bioinformatic tools specifically designed to genotype paralogous alleles including, the PacBio star-typer (Pangu) 48 for CYP2D6 and Paraphase 49 for other paralogous genes such as SMN1/SMN2, NCF1/NCF1B/NCF1C, PMS2 and CFC1/CFC1B . Lastly, the workflow consolidates all variant calls into standardized VCF files per sample (see methods).…”

Section: Resultsmentioning

confidence: 99%

Closing the gap: Solving complex medically relevant genes at scale

Mahmoud,

Harting,

Corbitt

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

Comprehending the mechanism behind human diseases with an established heritable component represents the forefront of personalized medicine. Nevertheless, numerous medically important genes are inaccurately represented in short-read sequencing data analysis due to their complexity and repetitiveness or the so-called "dark regions" of the human genome. The advent of PacBio as a long-read platform has provided new insights, yet HiFi whole-genome sequencing (WGS) cost remains frequently prohibitive. We introduce a targeted sequencing and analysis framework, Twist Alliance Dark Genes Panel (TADGP), designed to offer phased variants across 389 medically important yet complex autosomal genes. We highlight TADGP accuracy across eleven control samples and compare it to WGS. This demonstrates that TADGP achieves variant calling accuracy comparable to HiFi-WGS data, but at a fraction of the cost. Thus, enabling scalability and broad applicability for studying rare diseases or complementing previously sequenced samples to gain insights into these complex genes. TADGP revealed several candidate variants across all cases and provided insight into LPA diversity when tested on samples from rare disease and cardiovascular disease cohorts. In both cohorts, we identified novel variants affecting individual disease-associated genes (e.g., IKZF1, KCNE1). Nevertheless, the annotation of the variants across these 389 medically important genes remains challenging due to their underrepresentation in ClinVar and gnomAD. Consequently, we also offer an annotation resource to enhance the evaluation and prioritization of these variants. Overall, we can demonstrate that TADGP offers a cost-efficient and scalable approach to routinely assess the dark regions of the human genome with clinical relevance.

show abstract

Section: Resultsmentioning

confidence: 99%

Closing the gap: Solving complex medically relevant genes at scale

Mahmoud,

Harting,

Corbitt

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Extensive gene conversion and unequal crossing over can result in highly similar gene copies that can no longer be separated into different genes based on sequence alone. For example, SMN1 and SMN2 are different in sequence in Exons 7-8, but are indistinguishable in Exons 1-6 indicating that gene conversion is much more common in Exons 1-6 than in Exons 7-8 (Chen et al 2023). Thus, a principal component analysis (PCA) of variants in Exons 7-8 can differentiate SMN1 haplotypes from SMN2 haplotypes, but a PCA of variants in Exons 1-6 does not differentiate the genes (Figure S3).…”

Section: Resultsmentioning

confidence: 99%

“…Paraphase: HiFi-based caller for highly similar paralogous genes Paraphase is designed to work with both PacBio HiFi WGS and target enrichment data. Paraphase resolves a group of highly similar genes by extracting HiFi reads aligned to any member of the family, realigning them to the archetype gene, and phasing them into haplotypes, followed by variant calling on each haplotype (Chen et al 2023) (Figure 1A). Realigning all reads from all genes of the same gene family to one gene bypasses the error-prone process of aligning reads to multiple similar regions.…”

Section: Methodsmentioning

confidence: 99%

Genome-wide profiling of highly similar paralogous genes using HiFi sequencing

Chen,

Baker,

Dolzhenko

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

Variant calling is hindered in segmental duplications by sequence homology. We developed Paraphase, a HiFi-based informatics method that resolves highly similar genes by phasing all haplotypes of a gene family. We applied Paraphase to 160 long (>10 kb) segmental duplication regions across the human genome with high (>99%) sequence similarity, encoding 316 genes. Analysis across five ancestral populations revealed highly variable copy numbers of these regions. We identified 23 families with exceptionally low within-family diversity, where extensive gene conversion and unequal-crossing over have resulted in highly similar gene copies. Furthermore, our analysis of 36 trios identified 7de novoSNVs and 4de novogene conversion events, 2 of which are non-allelic. Finally, we summarized extensive genetic diversity in 9 medically relevant genes previously considered challenging to genotype. Paraphase provides a framework for resolving gene paralogs, enabling accurate testing in medically relevant genes and population-wide studies of previously inaccessible genes.

show abstract

“…Data from both amplicons were combined to achieve the highest accuracy for the prototype assay (>97%). Other long-read sequencing designs utilize only phased haplotype analysis either by hybrid capture (20) or multiple ultra-long (>26kb) amplicons on the PacBio SMRT platform (19). Robustness may be adversely affected by identical haplotypes without read depth normalized to endogenous and exogenous control amplicons.…”

Section: Discussionmentioning

confidence: 99%

A dual-mode targeted Nanopore sequencing assay for comprehensiveSMN1andSMN2variant analysis

Hall,

Alyafei,

Ramaswamy

et al. 2024

Preprint

View full text Add to dashboard Cite

Background Spinal Muscular Atrophy (SMA) is one of the most common recessive disorders for which several life-saving treatment options are currently available. It is essential to establish universal SMA screening and diagnostic programs using scalable, cost-effective and accessible platforms to accurately identify all variation types, which is complicated by homologousSMN1andSMN2genes. Methods We developed a dual-mode PCR-based target enrichment that generates 2.7 to 11.2 kb amplicons spanningSMN1andSMN2genes for any-length nanopore sequencing. We trained a variant calling model that utilizes paralog-specific sequences and read-depth data to accurately detect sequence and copy number variants specific to each gene. Results We present results from the development, optimization, and external evaluation of this assay using over 750 samples, including cell lines, residual presumed normal blood donors, and patients with knownSMN1andSMN2genotypes. The assay detects SNVs, indels, and CNVs with >98% accuracy across all sample sets, with a highly dynamic throughput range, relatively fast turnaround time, and limited hands-on-time. Together with the modest capital investment and consumable costs per sample, this assay can help increase access to SMA testing in low- and middle-income settings. Conclusion We describe a PCR/Nanopore sequencing assay and a customized analysis pipeline for the comprehensive and accurate detection of variation at the SMA locus and demonstrate its scalability, cost-effectiveness, and potential for the universal implementation of SMA screening and diagnostic programs. Human Genes SMN1survival of motor neuron 1, telomeric HGNC:11117SMN2survival of motor neuron 2, centromeric HGNC:11118CFTRCF transmembrane conductance regulator HGNC:1884

show abstract

Comprehensive SMN1 and SMN2 profiling for spinal muscular atrophy analysis using long-read PacBio HiFi sequencing

Cited by 32 publications

References 35 publications

Closing the gap: Solving complex medically relevant genes at scale

Closing the gap: Solving complex medically relevant genes at scale

Genome-wide profiling of highly similar paralogous genes using HiFi sequencing

A dual-mode targeted Nanopore sequencing assay for comprehensiveSMN1andSMN2variant analysis

Contact Info

Product

Resources

About