| 1. |
- Ameur, Adam, et al.
(författare)
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De Novo Assembly of Two Swedish Genomes Reveals Missing Segments from the Human GRCh38 Reference and Improves Variant Calling of Population-Scale Sequencing Data
- 2018
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Ingår i: Genes. - : MDPI AG. - 2073-4425 .- 2073-4425. ; 9:10
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Tidskriftsartikel (refereegranskat)abstract
- The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be assigned to chromosome Y, and large segments are also missing from GRCh38 at chromosomes 14, 17, and 21. Inclusion of NS into the GRCh38 reference radically improves the alignment and variant calling from short-read whole-genome sequencing data at several genomic loci. A re-analysis of a Swedish population-scale sequencing project yields > 75,000 putative novel single nucleotide variants (SNVs) and removes > 10,000 false positive SNV calls per individual, some of which are located in protein coding regions. Our results highlight that the GRCh38 reference is not yet complete and demonstrate that personal genome assemblies from local populations can improve the analysis of short-read whole-genome sequencing data.
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| 2. |
- Ameur, Adam, et al.
(författare)
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SweGen : a whole-genome data resource of genetic variability in a cross-section of the Swedish population
- 2017
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Ingår i: European Journal of Human Genetics. - : NATURE PUBLISHING GROUP. - 1018-4813 .- 1476-5438. ; 25:11, s. 1253-1260
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Tidskriftsartikel (refereegranskat)abstract
- Here we describe the SweGen data set, a comprehensive map of genetic variation in the Swedish population. These data represent a basic resource for clinical genetics laboratories as well as for sequencing-based association studies by providing information on genetic variant frequencies in a cohort that is well matched to national patient cohorts. To select samples for this study, we first examined the genetic structure of the Swedish population using high-density SNP-array data from a nation-wide cohort of over 10 000 Swedish-born individuals included in the Swedish Twin Registry. A total of 1000 individuals, reflecting a cross-section of the population and capturing the main genetic structure, were selected for whole-genome sequencing. Analysis pipelines were developed for automated alignment, variant calling and quality control of the sequencing data. This resulted in a genome-wide collection of aggregated variant frequencies in the Swedish population that we have made available to the scientific community through the website https://swefreq.nbis.se. A total of 29.2 million single-nucleotide variants and 3.8 million indels were detected in the 1000 samples, with 9.9 million of these variants not present in current databases. Each sample contributed with an average of 7199 individual-specific variants. In addition, an average of 8645 larger structural variants (SVs) were detected per individual, and we demonstrate that the population frequencies of these SVs can be used for efficient filtering analyses. Finally, our results show that the genetic diversity within Sweden is substantial compared with the diversity among continental European populations, underscoring the relevance of establishing a local reference data set.
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| 3. |
- Höijer, Ida, et al.
(författare)
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CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations
- 2022
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- CRISPR-Cas9 genome editing has potential to cure diseases without current treatments, but therapies must be safe. Here we show that CRISPR-Cas9 editing can introduce unintended mutations in vivo, which are passed on to the next generation. By editing fertilized zebrafish eggs using four guide RNAs selected for off-target activity in vitro, followed by long-read sequencing of DNA from >1100 larvae, juvenile and adult fish across two generations, we find that structural variants (SVs), i.e., insertions and deletions >= 50 bp, represent 6% of editing outcomes in founder larvae. These SVs occur both at on-target and off-target sites. Our results also illustrate that adult founder zebrafish are mosaic in their germ cells, and that 26% of their offspring carries an off-target mutation and 9% an SV. Hence, pre-testing for off-target activity and SVs using patient material is advisable in clinical applications, to reduce the risk of unanticipated effects with potentially large implications.
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| 4. |
- Höijer, Ida, et al.
(författare)
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Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing
- 2018
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Ingår i: Human Mutation. - : Hindawi Limited. - 1059-7794 .- 1098-1004. ; 39:9, s. 1262-1272
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Tidskriftsartikel (refereegranskat)abstract
- Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine-cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification-free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No-Amp Targeted sequencing) in combination with single molecule, real-time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification-free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No-Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR-based methods.
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| 5. |
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| 6. |
- Höijer, Ida
(författare)
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Targeted Long-read Sequencing : Development and Applications in Medical Genetics
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Targeted sequencing has the advantage of providing pinpointed DNA information, while costs and data-analysis efforts are reduced. If targeted sequencing is combined with single molecule long-read sequencing, it can become a powerful tool to investigate genomic regions traditionally difficult using the predominantly used short-read sequencing platforms, including repetitive regions and large structural variants.The aim of this thesis has been to develop and apply novel targeted long-read sequencing protocols to solve research questions of biomedical and clinical interest. In Paper I we utilized a new amplification-free targeted long-read sequencing method to study trinucleotide repeats in the huntingtin (HTT) gene, associated with Huntington’s disease. This method generated reads spanning the entire repeats, and we could accurately determine the repeat sizes in patient samples. Moreover, we could discover somatic variation of HTT repeat elements as a result of sequencing single, unamplified DNA molecules. In Paper II we present the Xdrop technology, a microfluidic-based system for targeted enrichment of large DNA molecules in droplets from low input samples. We applied the Xdrop technology to detect human papilloma virus 18 (HPV18) integration sites in the human genome of a cervical cancer cell line by targeting the virus genome. We also demonstrated its utility in detecting and phasing SNVs in the tumor suppressor gene TP53 in leukemia cells. In Paper III we employed targeted long-read sequencing to identify CRISPR-Cas9 off-target mutations in vitro with our two novel methods Nano-OTS and SMRT-OTS. Importantly, we were able to identify Cas9 cleavage sites in regions of the human genome that are difficult or impossible to assess using short-read sequencing. The aim of Paper IV was to investigate large structural variants (SVs) induced by CRISPR-Cas9 at on-target and off-target sites in genome edited zebrafish and their offspring. Nano-OTS was used to identify Cas9 off-target sites for four guide RNAs, which were also used for genome editing of fertilized fish eggs. Aided by long-read re-sequencing, we showed that Cas9 can induce large SVs at both on-target and off-target sites in vivo, and that these adverse variants can be passed on to the next generation.This thesis has highlighted a diversity of targeted long-read sequencing methods and some of their applications in medical genetics. We believe these methods could have an important place in future research and clinical diagnostics, and that the scope of their utility will be far beyond the applications demonstrated in this work.
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