Isaac: ultra-fast whole-genome secondary analysis on Illumina sequencing platforms

Raczy, Come; Petrovski, Roman; Saunders, Christopher T.; Chorny, Ilya; Kruglyak, Semyon; Margulies, Elliott H.; Chuang, Han‐Yu; Källberg, Morten; Kumar, Swathi Ashok; Liao, Arnold; Little, Kristina M.; Strömberg, Michael; Tanner, Stephen

doi:10.1093/bioinformatics/btt314

Cited by 303 publications

(282 citation statements)

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“…DNA libraries were subjected to WGS using an Illumina HiSeq X Ten Sequencer at Macrogen (30× to 40× ). The sequence file was aligned to the human reference genome hg19 from UCSC with the following mapping program and parameters using Isaac aligner 48 : base quality cutoff, 15; keep duplicate reads, yes; variable read length support, yes; realign gaps, no; and adaptor clipping, yes (adaptor: AGATCGGAAGAGC* , * GCTCTTCCGATCT).…”

Section: Article Researchmentioning

confidence: 99%

“…WGS was performed using an Illumina HiSeq X Ten sequencer with a sequencing depth of 30× to 40× (Macrogen, South Korea). Sequences from each blastomere were processed to obtain total variants (lane 1 in Extended Data Table 6) using the Isaac variant calling program 48 . Annotated variants, including dbSNPs and all novel SNPs (substitution changes), were filtered out, and novel indel sites were identified (lane 2 in Extended Data Table 6).…”

Section: Article Researchmentioning

confidence: 99%

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Correction of a pathogenic gene mutation in human embryos

Martí-Gutiérrez

Park

et al. 2017

Nature

814

540

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More than 10,000 monogenic inherited disorders have been identified, affecting millions of people worldwide. Among these are autosomal dominant mutations, where inheritance of a single copy of a defective gene can result in clinical symptoms. Genes in which dominant mutations manifest as late-onset adult disorders include BRCA1 and BRCA2, which are associated with a high risk of breast and ovarian cancers 1 , and MYBPC3, mutation of which causes hypertrophic cardiomyopathy (HCM) 2 . Because of their delayed manifestation, these mutations escape natural selection and are often transmitted to the next generation. Consequently, the frequency of some of these founder mutations in particular human populations is very high. For example, the MYBPC3 mutation is found at frequencies ranging from 2% to 8% 3 in major Indian populations, and the estimated frequency of both BRCA1 and BRCA2 mutations among Ashkenazi Jews exceeds 2% 4 .HCM is a myocardial disease characterized by left ventricular hypertrophy, myofibrillar disarray and myocardial stiffness; it has an estimated prevalence of 1:500 in adults 5 and manifests clinically with heart failure. HCM is the commonest cause of sudden death in otherwise healthy young athletes. HCM, while not a uniformly fatal condition, has a tremendous impact on the lives of individuals, including physiological (heart failure and arrhythmias), psychological (limited activity and fear of sudden death), and genealogical concerns. MYBPC3 mutations account for approximately 40% of all genetic defects causing HCM and are also responsible for a large fraction of other inherited cardiomyopathies, including dilated cardiomyopathy and left ventricular non-compaction 6 . MYBPC3 encodes the thick filament-associated cardiac myosin-binding protein C (cMyBP-C), a signalling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of both contraction and relaxation 2 .Current treatment options for HCM provide mostly symptomatic relief without addressing the genetic cause of the disease. Thus, the development of novel strategies to prevent germline transmission of founder mutations is desirable. One approach for preventing second-generation transmission is preimplantation genetic diagnosis (PGD) followed by selection of non-mutant embryos for transfer in the context of an in vitro fertilization (IVF) cycle. When only one parent carries a heterozygous mutation, 50% of the embryos should be mutationfree and available for transfer, while the remaining carrier embryos are discarded. Gene correction would rescue mutant embryos, increase the number of embryos available for transfer and ultimately improve pregnancy rates.Recent developments in precise genome-editing techniques and their successful applications in animal models have provided an option for correcting human germline mutations. In particular, CRISPR-Cas9 is a versatile tool for recognizing specific genomic sequences and inducing DSBs 7-10 . DSBs are then resolved by endogenous DNA repair mechanisms, prefer...

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Section: Article Researchmentioning

confidence: 99%

Section: Article Researchmentioning

confidence: 99%

Correction of a pathogenic gene mutation in human embryos

Martí-Gutiérrez

Park

et al. 2017

Nature

814

540

View full text Add to dashboard Cite

show abstract

“…WGS is performed at a sequencing depth of 30× to 40×. We used Isaac aligner to align the sequence file to the human reference genome hg19 (Raczy et al 2013) or GRCh38 (Cunningham et al 2015) with the following mapping program and parameters: base quality cutoff, 15; keep duplicate reads, yes; variable read length support, yes; realign gaps, no; and adaptor clipping, yes (adaptor AGATCGGAAGAGC * , * GCTCTTCCGATCT). DNA cleavage sites were identified computationally using a cleavage scoring system described in Supplemental Figure 1.…”

Section: Whole-genome and Digenome Sequencingmentioning

confidence: 99%

Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

et al. 2016

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We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on-and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many falsepositive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.

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“…To identify these variants, the Illumina team will use the company's Isaac workflow, an open-source computational genomealignment and variant-calling tool 3 . Then, to discover which of each genome's hundreds of thousands of variants have a role in the individual's disease, Genomics England will gather and analyse the sequences and variants from all 100,000 genomes at its secure data centre in Corsham, UK.…”

Section: Industry Partnershipmentioning

confidence: 99%