ORCID Profile
0000-0003-2494-8587
Current Organisations
University of Sydney
,
Department of Health
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Publisher: Springer Science and Business Media LLC
Date: 12-01-2022
DOI: 10.1186/S13059-021-02579-6
Abstract: Next-generation sequencing (NGS) can identify mutations in the human genome that cause disease and has been widely adopted in clinical diagnosis. However, the human genome contains many polymorphic, low-complexity, and repetitive regions that are difficult to sequence and analyze. Despite their difficulty, these regions include many clinically important sequences that can inform the treatment of human diseases and improve the diagnostic yield of NGS. To evaluate the accuracy by which these difficult regions are analyzed with NGS, we built an in silico decoy chromosome, along with corresponding synthetic DNA reference controls, that encode difficult and clinically important human genome regions, including repeats, microsatellites, HLA genes, and immune receptors. These controls provide a known ground-truth reference against which to measure the performance of erse sequencing technologies, reagents, and bioinformatic tools. Using this approach, we provide a comprehensive evaluation of short- and long-read sequencing instruments, library preparation methods, and software tools and identify the errors and systematic bias that confound our resolution of these remaining difficult regions. This study provides an analytical validation of diagnosis using NGS in difficult regions of the human genome and highlights the challenges that remain to resolve these difficult regions.
Publisher: Springer Science and Business Media LLC
Date: 22-03-2019
DOI: 10.1038/S41467-019-09272-0
Abstract: Chirality is a property describing any object that is inequivalent to its mirror image. Due to its 5′–3′ directionality, a DNA sequence is distinct from a mirrored sequence arranged in reverse nucleotide-order, and is therefore chiral. A given sequence and its opposing chiral partner sequence share many properties, such as nucleotide composition and sequence entropy. Here we demonstrate that chiral DNA sequence pairs also perform equivalently during molecular and bioinformatic techniques that underpin genetic analysis, including PCR lification, hybridization, whole-genome, target-enriched and nanopore sequencing, sequence alignment and variant detection. Given these shared properties, synthetic DNA sequences mirroring clinically relevant or analytically challenging regions of the human genome are ideal controls for clinical genomics. The addition of synthetic chiral sequences (sequins) to patient tumor s les can prevent false-positive and false-negative mutation detection to improve diagnosis. Accordingly, we propose that sequins can fulfill the need for commutable internal controls in precision medicine.
Publisher: Cold Spring Harbor Laboratory
Date: 31-08-2018
DOI: 10.1101/404285
Abstract: Chirality is a geometric property describing any object that is inequivalent to a mirror image of itself. Due to its 5’-3’ directionality, a DNA sequence is distinct from a mirrored sequence arranged in reverse nucleotide order, and is therefore chiral. A given sequence and its opposing chiral partner sequence share many properties, such as nucleotide composition and sequence entropy. Here we demonstrate that chiral DNA sequence pairs also perform equivalently during molecular and bioinformatic techniques that underpin modern genetic analysis, including PCR lification, hybridization, whole-genome, target-enriched and nanopore sequencing, sequence alignment and variant detection. Given these shared properties, synthetic DNA sequences that directly mirror clinically relevant and/or analytically challenging regions of the human genome are ideal reference standards for clinical genomics. We show how the addition of chiral DNA standards to patient tumor s les can prevent false-positive and false-negative mutation detection and, thereby, improve diagnosis. Accordingly, we propose that chiral DNA standards can fulfill the unmet need for commutable internal reference standards in precision medicine.
Publisher: ACM
Date: 30-01-2023
Publisher: Springer Science and Business Media LLC
Date: 19-06-2019
DOI: 10.1038/S41596-019-0175-1
Abstract: Next-generation sequencing (NGS) has been widely adopted to identify genetic variants and investigate their association with disease. However, the analysis of sequencing data remains challenging because of the complexity of human genetic variation and confounding errors introduced during library preparation, sequencing and analysis. We have developed a set of synthetic DNA spike-ins-termed 'sequins' (sequencing spike-ins)-that are directly added to DNA s les before library preparation. Sequins can be used to measure technical biases and to act as internal quantitative and qualitative controls throughout the sequencing workflow. This step-by-step protocol explains the use of sequins for both whole-genome and targeted sequencing of the human genome. This includes instructions regarding the dilution and addition of sequins to human DNA s les, followed by the bioinformatic steps required to separate sequin- and s le-derived sequencing reads and to evaluate the diagnostic performance of the assay. These practical guidelines are accompanied by a broader discussion of the conceptual and statistical principles that underpin the design of sequin standards. This protocol is suitable for users with standard laboratory and bioinformatic experience. The laboratory steps require ~1-4 d and the bioinformatic steps (which can be performed with the provided ex le data files) take an additional day.
No related grants have been discovered for Christopher Barker.