Next-generation sequencing as a tool for breakpoint analysis in rearrangements of the globin gene clusters
| Autor: | David Brawand, Frances Smith, Barnaby Clark, Claire Shooter, Swee Lay Thein |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Clinical Biochemistry Biology Deep sequencing DNA sequencing Chromosome Breakpoints 03 medical and health sciences symbols.namesake 0302 clinical medicine Humans Exome Gene Rearrangement Sanger sequencing Genetics Massive parallel sequencing Genome Human Biochemistry (medical) Breakpoint High-Throughput Nucleotide Sequencing Hematology General Medicine Gene rearrangement Amplicon Globins 030104 developmental biology Multigene Family 030220 oncology & carcinogenesis symbols Thalassemia |
| Zdroj: | International Journal of Laboratory Hematology. 39:111-120 |
| ISSN: | 1751-5521 |
| DOI: | 10.1111/ijlh.12680 |
| Popis: | Introduction Next-generation sequencing (NGS), now embedded within genomic laboratories, is well suited to the detection of small sequence changes but is less well adapt for detecting structural variants (SV), mainly due to the relatively short sequence reads. Of the available target enrichment methods, bait capture or whole-genome sequencing appears better suited to detecting SV as there is less PCR amplification and is therefore more representative of the genome being sequenced. Material and methods In 2015, we described the first inversion/deletion causing eγδβ- thalassemia using an NGS approach, with base-pair resolution. Bioinformatic processing of the sequencing data was manual and time-consuming. The methodology relied on detecting the presence or absence of the SV by assessing sequence coverage and then mapping the deletion by capturing and sequencing breakpoint spanning reads (split reads). In the period between developing more automated analytical methods, we identified the first duplication of the entire beta globin cluster. Results Detecting the presence of the SV is reliable but capturing the breakpoint spanning reads is challenging. Confirmation by Sanger sequencing a breakpoint spanning amplicon has confirmed the NGS results in all cases. Conclusions We have now streamlined and automated the bioinformatic approach using Exome Depth to assess sequence coverage and Delly to detect split and discordant reads. The combined NGS and bioinformatic strategy has proven to be highly successful and applicable to routine diagnostics. |
| Databáze: | OpenAIRE |
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