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Comparative Study
. 2020 Mar;8(3):e1114.
doi: 10.1002/mgg3.1114. Epub 2020 Jan 27.

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

Kévin Uguen #  1   2 , Claire Jubin #  3   4 , Yannis Duffourd  5 , Claire Bardel  6   7 , Valérie Malan  8 , Jean-Michel Dupont  9 , Laila El Khattabi  9 , Nicolas Chatron  2 , Antonio Vitobello  5   10 , Pierre-Antoine Rollat-Farnier  6 , Céline Baulard  3   4 , Marc Lelorch  8 , Aurélie Leduc  3   4 , Emilie Tisserant  5 , Frédéric Tran Mau-Them  5   10 , Vincent Danjean  11 , Marc Delepine  3   4 , Marianne Till  2 , Vincent Meyer  3   4 , Stanislas Lyonnet  12 , Anne-Laure Mosca-Boidron  5   13 , Julien Thevenon  14 , Laurence Faivre  5   15 , Christel Thauvin-Robinet  5   15 , Caroline Schluth-Bolard  2 , Anne Boland  3   4 , Robert Olaso  3   4 , Patrick Callier  5   13 , Serge Romana  8 , Jean-François Deleuze #  3   4 , Damien Sanlaville #  2
Affiliations
Comparative Study

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

Kévin Uguen et al. Mol Genet Genomic Med. 2020 Mar.

Abstract

Background: Structural variants (SVs) include copy number variants (CNVs) and apparently balanced chromosomal rearrangements (ABCRs). Genome sequencing (GS) enables SV detection at base-pair resolution, but the use of short-read sequencing is limited by repetitive sequences, and long-read approaches are not yet validated for diagnosis. Recently, 10X Genomics proposed Chromium, a technology providing linked-reads to reconstruct long DNA fragments and which could represent a good alternative. No study has compared short-read to linked-read technologies to detect SVs in a constitutional diagnostic setting yet. The aim of this work was to determine whether the 10X Genomics technology enables better detection and comprehension of SVs than short-read WGS.

Methods: We included 13 patients carrying various SVs. Whole genome analyses were performed using paired-end HiSeq X sequencing with (linked-read strategy) or without (short-read strategy) Chromium library preparation. Two different bioinformatic pipelines were used: Variants are called using BreakDancer for short-read strategy and LongRanger for long-read strategy. Variant interpretations were first blinded.

Results: The short-read strategy allowed diagnosis of known SV in 10/13 patients. After unblinding, the linked-read strategy identified 10/13 SVs, including one (patient 7) missed by the short-read strategy.

Conclusion: In conclusion, regarding the results of this study, 10X Genomics solution did not improve the detection and characterization of SV.

Keywords: 10X Genomics: Illumina; bioinformatics; genome sequencing; structural variants.

PubMed Disclaimer

Conflict of interest statement

10X company paid half of the necessary reagents for the preparation of the linked‐read library.

Figures

Figure 1
Figure 1
Study workflow. All the patients were analyzed with both strategies. The three first steps were mandatory. The last step, after unblinding, was performed only if the previous analysis was not able to find the expected SV
Figure 2
Figure 2
Patient 2: SV representation and results from the linked‐read strategy. (A). The derivative chromosome from t(9;13) is represented here, with the normal chromosomes of patient 2. The distal region of the short arm of the chromosome 9 is deleted, and a 900 Kb region of the chromosome 9 in the vicinity of the breakpoint is duplicated. The distal part of the long arm of the chromosome 13 is duplicated. (B) IGV visualization of the breakpoint located on chromosome 13 shows that there is a difference in depth from either side of the breakpoint (represented by the black vertical line). (C) A screen shot from the Loupe visualization. Shown are linear (top left and right panels) and matrix (bottom left and right panels) representations at the breakpoint intervals. The left panels show the coordinates of the two breakpoints from chromosome 9 and 13 as well as the translocation site (pinpointed by the black arrow). The right panel displays a focus on the chromosome 13 breakpoint showing a mild increase in read depth for the distal segment, corresponding to the duplication in chromosome 13 (red arrow)
Figure 3
Figure 3
Patients 4 and 5. (A) Circos plot of the chromothripsis of patient 4. We note that there is a certain clustering of the breakpoints on chromosomes 4 and 14. (B) Chromosome representation of the CNVs from patient 5. The left panel represents the normal chromosome. The breakpoints of the proximal inserted segment and those of the distal deleted segment are indicated. The right panel represents the rearranged chromosome with the 100 kb proximal duplicated segment being inserted between the breakpoints of the 50 kb distal deletion
Figure 4
Figure 4
Patient 7: SV representation and results. (A). The (X;1) reciprocal translocation is represented here, with the coordinates of the two breakpoints. Chromosome 1 is colored in orange and chromosome X in blue. (B) IGV visualization and UCSC genome browser show that the breakpoint on chromosome X (indicated by the black dashed line) disrupts the CLCN5 gene and is located in a LINE sequence. (C) Results of the specific PCR amplification of the two fusion points at both derivative chromosomes (der1 and derX) and a control locus (on the ATP1A3 gene). NC = negative control corresponding to DNA from an individual who does not have the translocation
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