Genome sequencing in cytogenetics: Comparison of short‐read and linked‐read approaches for germline structural variant detection and characterization

Uguen, Kévin; Jubin, Claire; Duffourd, Yannis; Bardel, Claire; Malan, Valérie; Dupont, Jean‐Michel; Khattabi, Laïla El; Chatron, Nicolas; Vitobello, Antonio; Rollat‐Farnier, Pierre‐Antoine; Baulard, Céline; Lelorc’h, Marc; Leduc, Aurélie; Tisserant, Émilie; Mau‐Them, Frédéric Tran; Danjean, Vincent; Délépine, Marc; Till, Marianne; Meyer, Vincent; Lyonnet, Stanislas; Mosca-Boidron, Anne-Laure; Thévenon, Julien; Faivre, Laurence; Thauvin‐Robinet, Christel; Schluth-Bolard, Caroline; Boland, Anne; Olaso, Robert; Callier, Patrick; Romana, Serge; Deleuze, Jean‐François; Sanlaville, Damien

doi:10.1002/mgg3.1114

Cited by 10 publications

(13 citation statements)

References 43 publications

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“…Notably, beside short read standard WGS we also performed linked‐read sequencing (10x genomics) to see whether this technology could overcome any of the technical challenges encountered through WGS and analysis (Supplementary Figure 8). Consistent with other reports, we did not find any significant advantage and it is now discontinued 22 . Interestingly sequencing of the insertion point revealed the exact same sequence for all patients and the genotyping of some individuals revealed a common shared haplotype.…”

Section: Discussionsupporting

confidence: 91%

A BBS1 SVA F retrotransposon insertion is a frequent cause of Bardet‐Biedl syndrome

et al. 2020

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Bardet‐Biedl syndrome (BBS) is a ciliopathy characterized by retinitis pigmentosa, obesity, polydactyly, cognitive impairment and renal failure. Pathogenic variants in 24 genes account for the molecular basis of >80% of cases. Toward saturated discovery of the mutational basis of the disorder, we carefully explored our cohorts and identified a hominid‐specific SINE‐R/VNTR/Alu type F (SVA‐F) insertion in exon 13 of BBS1 in eight families. In six families, the repeat insertion was found in trans with c.1169 T > G, p.Met390Arg and in two families the insertion was found in addition to other recessive BBS loci. Whole genome sequencing, de novo assembly and SNP array analysis were performed to characterize the genomic event. This insertion is extremely rare in the general population (found in 8 alleles of 8 BBS cases but not in >10 800 control individuals from gnomAD‐SV) and due to a founder effect. Its 2435 bp sequence contains hallmarks of LINE1 mediated retrotransposition. Functional studies with patient‐derived cell lines confirmed that the BBS1 SVA‐F is deleterious as evidenced by a significant depletion of both mRNA and protein levels. Such findings highlight the importance of dedicated bioinformatics pipelines to identify all types of variation.

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Section: Discussionsupporting

confidence: 91%

A BBS1 SVA F retrotransposon insertion is a frequent cause of Bardet‐Biedl syndrome

et al. 2020

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“…It uses microfluidics partitions to produce droplet particles and avoids any fragmentation, making it ideal for sequencing high molecular weight DNA of 50 kbs or higher. More importantly, the linked-read method allows mapping the reads from the repetitive regions where SV breakpoints are often located [ 32 , 33 ]. Recent studies have demonstrated the utility of the linked-read method for detecting complex SVs in a variety of samples [ 34 , 35 , 36 ].…”

Section: Introductionmentioning

confidence: 99%

“…The study reported that linked-read sequencing produced scalable and comprehensive information on SVs as compared to short-read sequencing. Later, Uguen et al compared the linked-read method with short read-based WGS to detect SVs [ 33 ]. The study reported that linked-read sequencing could not improve the detection or characterization of SVs.…”

Section: Introductionmentioning

confidence: 99%

Comparison of Structural and Short Variants Detected by Linked-Read and Whole-Exome Sequencing in Multiple Myeloma

Kumar

Adhikari

Kankainen

et al. 2021

Cancers

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Linked-read sequencing was developed to aid the detection of large structural variants (SVs) from short-read sequencing efforts. We performed a systematic evaluation to determine if linked-read exome sequencing provides more comprehensive and clinically relevant information than whole-exome sequencing (WES) when applied to the same set of multiple myeloma patient samples. We report that linked-read sequencing detected a higher number of SVs (n = 18,455) than WES (n = 4065). However, linked-read predictions were dominated by inversions (92.4%), leading to poor detection of other types of SVs. In contrast, WES detected 56.3% deletions, 32.6% insertions, 6.7% translocations, 3.3% duplications and 1.2% inversions. Surprisingly, the quantitative performance assessment suggested a higher performance for WES (AUC = 0.791) compared to linked-read sequencing (AUC = 0.766) for detecting clinically validated cytogenetic alterations. We also found that linked-read sequencing detected more short variants (n = 704) compared to WES (n = 109). WES detected somatic mutations in all MM-related genes while linked-read sequencing failed to detect certain mutations. The comparison of somatic mutations detected using linked-read, WES and RNA-seq revealed that WES and RNA-seq detected more mutations than linked-read sequencing. These data indicate that WES outperforms and is more efficient than linked-read sequencing for detecting clinically relevant SVs and MM-specific short variants.

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“…While linked-reads (e.g. 10x Genomics) seem promising, especially for cases in repetitive elements, the technique and detection tools need further development [17].…”

Section: Introductionmentioning

confidence: 99%

Using short read sequencing to characterise balanced reciprocal translocations in pigs

et al. 2020

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Background: A balanced constitutional reciprocal translocation (RT) is a mutual exchange of terminal segments of two non-homologous chromosomes without any loss or gain of DNA in germline cells. Carriers of balanced RTs are viable individuals with no apparent phenotypical consequences. These animals produce, however, unbalanced gametes and show therefore reduced fertility and offspring with congenital abnormalities. This cytogenetic abnormality is usually detected using chromosome staining techniques. The aim of this study was to test the possibilities of using paired end short read sequencing for detection of balanced RTs in boars and investigate their breakpoints and junctions. Results: Balanced RTs were recovered in a blinded analysis, using structural variant calling software DELLY, in 6 of the 7 carriers with 30 fold short read paired end sequencing. In 15 non-carriers we did not detect any RTs. Reducing the coverage to 20 fold, 15 fold and 10 fold showed that at least 20 fold coverage is required to obtain good results. One RT was not detected using the blind screening, however, a highly likely RT was discovered after unblinding. This RT was located in a repetitive region, showing the limitations of short read sequence data. The detailed analysis of the breakpoints and junctions suggested three junctions showing microhomology, three junctions with blunt-end ligation, and three micro-insertions at the breakpoint junctions. The RTs detected also showed to disrupt genes. Conclusions: We conclude that paired end short read sequence data can be used to detect and characterize balanced reciprocal translocations, if sequencing depth is at least 20 fold coverage. However, translocations in repetitive areas may require large fragments or even long read sequence data.

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Genome sequencing in cytogenetics: Comparison of short‐read and linked‐read approaches for germline structural variant detection and characterization

Cited by 10 publications

References 43 publications

A BBS1 SVA F retrotransposon insertion is a frequent cause of Bardet‐Biedl syndrome

A BBS1 SVA F retrotransposon insertion is a frequent cause of Bardet‐Biedl syndrome

Comparison of Structural and Short Variants Detected by Linked-Read and Whole-Exome Sequencing in Multiple Myeloma

Using short read sequencing to characterise balanced reciprocal translocations in pigs

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