Dataset Information

Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.

ABSTRACT: Leukemias are genetically heterogeneous and diagnostics therefore includes various standard-of-care (SOC) techniques, including karyotyping, SNP-array and FISH. Optical genome mapping (OGM) may replace these as it detects different types of structural aberrations simultaneously and additionally detects much smaller aberrations (500 bp vs 5-10 Mb with karyotyping). However, its resolution may still be too low to define clinical relevance of aberrations when they are located between two OGM labels or when labels are not distinct enough. Here, we test the potential of Cas9-directed long-read sequencing (LRS) as an additional technique to resolve such potentially relevant new findings. From an internal Bionano implementation study we selected ten OGM calls that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION/GridION device. We could confirm all aberrations and, importantly, the actual breakpoints of the OGM calls were located between 0.2 and 5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. Our results suggest that Cas9-directed LRS can be a relevant and flexible secondary technique in diagnostic workflows including OGM.

SUBMITTER: de Boer EN

PROVIDER: S-EPMC11009395 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.

de Boer Eddy N EN Vroom Vincent V Scheper Arjen J AJ Johansson Lennart F LF Bosscher Laura L Rietema Nettie N Commandeur-Jan Sabrina Z SZ Knoers Nine V A M NVAM Sikkema-Raddatz Birgit B van den Berg Eva E van Diemen Cleo C CC

Scientific reports 20240412 1

Leukemias are genetically heterogeneous and diagnostics therefore includes various standard-of-care (SOC) techniques, including karyotyping, SNP-array and FISH. Optical genome mapping (OGM) may replace these as it detects different types of structural aberrations simultaneously and additionally detects much smaller aberrations (500 bp vs 5-10 Mb with karyotyping). However, its resolution may still be too low to define clinical relevance of aberrations when they are located between two OGM labels ...[more]

PMID: 38605095

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Project description:BackgroundGenomic methods for identifying causative variants for trait loci applicable to a wide range of germplasm are required for plant biologists and breeders to understand the genetic control of trait variation.ResultsWe implemented Cas9-targeted sequencing for fine-mapping in apple, a method combining CRISPR-Cas9 targeted cleavage of a region of interest, followed by enrichment and long-read sequencing using the Oxford Nanopore Technology (ONT). We demonstrated the capability of this methodology to specifically cleave and enrich a plant genomic locus spanning 8 kb. The repeated mini-satellite motif located upstream of the Malus × domestica (apple) MYB10 transcription factor gene, causing red fruit colouration when present in a heterozygous state, was our exemplar to demonstrate the efficiency of this method: it contains a genomic region with a long structural variant normally ignored by short-read sequencing technologiesCleavage specificity of the guide RNAs was demonstrated using polymerase chain reaction products, before using them to specify cleavage of high molecular weight apple DNA. An enriched library was subsequently prepared and sequenced using an ONT MinION flow cell (R.9.4.1). Of the 7,056 ONT reads base-called using both Albacore2 (v2.3.4) and Guppy (v3.2.4), with a median length of 9.78 and 9.89 kb, respectively, 85.35 and 91.38%, aligned to the reference apple genome. Of the aligned reads, 2.98 and 3.04% were on-target with read depths of 180 × and 196 × for Albacore2 and Guppy, respectively, and only five genomic loci were off-target with read depth greater than 25 × , which demonstrated the efficiency of the enrichment method and specificity of the CRISPR-Cas9 cleavage.ConclusionsWe demonstrated that this method can isolate and resolve single-nucleotide and structural variants at the haplotype level in plant genomic regions. The combination of CRISPR-Cas9 target enrichment and ONT sequencing provides a more efficient technology for fine-mapping loci than genome-walking approaches.

Project description:BackgroundHigh sequence identity between segmental duplications (SDs) can facilitate copy number variants (CNVs) via non-allelic homologous recombination (NAHR). These CNVs are one of the fundamental causes of genomic disorders such as the 3q29 deletion syndrome (del3q29S). There are 21 protein-coding genes lost or gained as a result of such recurrent 1.6-Mbp deletions or duplications, respectively, in the 3q29 locus. While NAHR plays a role in CNV occurrence, the factors that increase the risk of NAHR at this particular locus are not well understood.MethodsWe employed an optical genome mapping technique to characterize the 3q29 locus in 161 unaffected individuals, 16 probands with del3q29S and their parents, and 2 probands with the 3q29 duplication syndrome (dup3q29S). Long-read sequencing-based haplotype resolved de novo assemblies from 44 unaffected individuals, and 1 trio was used for orthogonal validation of haplotypes and deletion breakpoints.ResultsIn total, we discovered 34 haplotypes, of which 19 were novel haplotypes. Among these 19 novel haplotypes, 18 were detected in unaffected individuals, while 1 novel haplotype was detected on the parent-of-origin chromosome of a proband with the del3q29S. Phased assemblies from 44 unaffected individuals enabled the orthogonal validation of 20 haplotypes. In 89% (16/18) of the probands, breakpoints were confined to paralogous copies of a 20-kbp segment within the 3q29 SDs. In one del3q29S proband, the breakpoint was confined to a 374-bp region using long-read sequencing. Furthermore, we categorized del3q29S cases into three classes and dup3q29S cases into two classes based on breakpoints. Finally, we found no evidence of inversions in parent-of-origin chromosomes.ConclusionsWe have generated the most comprehensive haplotype map for the 3q29 locus using unaffected individuals, probands with del3q29S or dup3q29S, and available parents, and also determined the deletion breakpoint to be within a 374-bp region in one proband with del3q29S. These results should provide a better understanding of the underlying genetic architecture that contributes to the etiology of del3q29S and dup3q29S.

Dataset Information

Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.

Publications

Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.

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