Diagnostic applications of next generation sequencing: working towards quality standards

Over the past 6 years, next generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. All major NGS technology companies providing commercially available instruments (Roche 454, Illumina, Life Technologies) have recently marketed bench top sequencing instruments with lower throughput and shorter run times, thereby broadening the applications of NGS and opening the technology to the potential use for clinical diagnostics. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry and data analysis need to be overcome. To facilitate the implementation of NGS as a routine method in molecular diagnostics, consistent quality standards need to be developed. Here the authors give an overview of the current standards in protocols and workflows and discuss possible approaches to define quality criteria for NGS in molecular genetic diagnostics

Bioinformatics and computational tools for next-generation sequencing analysis in clinical genetics

Clinical genetics has an important role in the healthcare system to provide a definitive diagnosis for many rare syndromes. It also can have an influence over genetics prevention, disease prognosis and assisting the selection of the best options of care/treatment for patients. Next-generation sequencing (NGS) has transformed clinical genetics making possible to analyze hundreds of genes at an unprecedented speed and at a lower price when comparing to conventional Sanger sequencing. Despite the growing literature concerning NGS in a clinical setting, this review aims to fill the gap that exists among (bio)informaticians, molecular geneticists and clinicians, by presenting a general overview of the NGS technology and workflow. First, we will review the current NGS platforms, focusing on the two main platforms Illumina and Ion Torrent, and discussing the major strong points and weaknesses intrinsic to each platform. Next, the NGS analytical bioinformatic pipelines are dissected, giving some emphasis to the algorithms commonly used to generate process data and to analyze sequence variants. Finally, the main challenges around NGS bioinformatics are placed in perspective for future developments. Even with the huge achievements made in NGS technology and bioinformatics, further improvements in bioinformatic algorithms are still required to deal with complex and genetically heterogeneous disorders

VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research

Accurate variant calling in next generation sequencing (NGS) is critical to understand cancer genomes better. Here we present VarDict, a novel and versatile variant caller for both DNA- and RNA-sequencing data. VarDict simultaneously calls SNV, MNV, InDels, complex and structural variants, expanding the detected genetic driver landscape of tumors. It performs local realignments on the fly for more accurate allele frequency estimation. VarDict performance scales linearly to sequencing depth, enabling ultra-deep sequencing used to explore tumor evolution or detect tumor DNA circulating in blood. In addition, VarDict performs amplicon aware variant calling for polymerase chain reaction (PCR)-based targeted sequencing often used in diagnostic settings, and is able to detect PCR artifacts. Finally, VarDict also detects differences in somatic and loss of heterozygosity variants between paired samples. VarDict reprocessing of The Cancer Genome Atlas (TCGA) Lung Adenocarcinoma dataset called known driver mutations in KRAS, EGFR, BRAF, PIK3CA and MET in 16% more patients than previously published variant calls. We believe VarDict will greatly facilitate application of NGS in clinical cancer research

From cheek swabs to consensus sequences : an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

Background: Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users. Results: Here we present an ‘A to Z’ protocol for obtaining complete human mitochondrial (mtDNA) genomes – from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling). Conclusions: All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual ‘modules’ can be swapped out to suit available resources

Sequence-Based Mapping and Genome Editing Reveal Mutations in Stickleback Hps5 Cause Oculocutaneous Albinism and the casper Phenotype.

Here, we present and characterize the spontaneous X-linked recessive mutation casper, which causes oculocutaneous albinism in threespine sticklebacks (Gasterosteus aculeatus). In humans, Hermansky-Pudlak syndrome results in pigmentation defects due to disrupted formation of the melanin-containing lysosomal-related organelle (LRO), the melanosome. casper mutants display not only reduced pigmentation of melanosomes in melanophores, but also reductions in the iridescent silver color from iridophores, while the yellow pigmentation from xanthophores appears unaffected. We mapped casper using high-throughput sequencing of genomic DNA from bulked casper mutants to a region of the stickleback X chromosome (chromosome 19) near the stickleback ortholog of Hermansky-Pudlak syndrome 5 (Hps5). casper mutants have an insertion of a single nucleotide in the sixth exon of Hps5, predicted to generate an early frameshift. Genome editing using CRISPR/Cas9 induced lesions in Hps5 and phenocopied the casper mutation. Injecting single or paired Hps5 guide RNAs revealed higher incidences of genomic deletions from paired guide RNAs compared to single gRNAs. Stickleback Hps5 provides a genetic system where a hemizygous locus in XY males and a diploid locus in XX females can be used to generate an easily scored visible phenotype, facilitating quantitative studies of different genome editing approaches. Lastly, we show the ability to better visualize patterns of fluorescent transgenic reporters in Hps5 mutant fish. Thus, Hps5 mutations present an opportunity to study pigmented LROs in the emerging stickleback model system, as well as a tool to aid in assaying genome editing and visualizing enhancer activity in transgenic fish

Next generation sequencing in cancer: opportunities and challenges for precision cancer medicine

Over the past decade, testing the genes of patients and their specific cancer types has become standardized practice in medical oncology since somatic mutations, changes in gene expression and epigenetic modifications are all hallmarks of cancer. However, while cancer genetic assessment has been limited to single biomarkers to guide the use of therapies, improvements in nucleic acid sequencing technologies and implementation of different genome analysis tools have enabled clinicians to detect these genomic alterations and identify functional and disease-associated genomic variants. Next-generation sequencing (NGS) technologies have provided clues about therapeutic targets and genomic markers for novel clinical applications when standard therapy has failed. While Sanger sequencing, an accurate and sensitive approach, allows for the identification of potential novel variants, it is however limited by the single amplicon being interrogated. Similarly, quantitative and qualitative profiling of gene expression changes also represents a challenge for the cancer field. Both RT-PCR and microarrays are efficient approaches, but are limited to the genes present on the array or being assayed. This leaves vast swaths of the transcriptome, including non-coding RNAs and other features, unexplored. With the advent of the ability to collect and analyze genomic sequence data in a timely fashion and at an ever-decreasing cost, many of these limitations have been overcome and are being incorporated into cancer research and diagnostics giving patients and clinicians new hope for targeted and personalized treatment. Below we highlight the various applications of next-generation sequencing in precision cancer medicine

Application of hybrid algorithms and Explainable Artificial Intelligence ingenomic sequencing

[EN]DNA sequencing is one of the fields that has advanced the most in recent years within clinical genetics and human biology. However, the large amount of data generated through next generation sequencing (NGS) techniques requires advanced data analysis processes that are sometimes complex and beyond the capabilities of clinical staff. Therefore, this work aims to shed light on the possibilities of applying hybrid algorithms and explainable artificial intelligence (XAI) to data obtained through NGS. The suitability of each architecture will be evaluated phase by phase in order to offer final recommendations that allow implementation in clinical sequencing workflow

Landscape of standing variation for tandem duplications in Drosophila yakuba and Drosophila simulans

We have used whole genome paired-end Illumina sequence data to identify tandem duplications in 20 isofemale lines of D. yakuba, and 20 isofemale lines of D. simulans and performed genome wide validation with PacBio long molecule sequencing. We identify 1,415 tandem duplications that are segregating in D. yakuba as well as 975 duplications in D. simulans, indicating greater variation in D. yakuba. Additionally, we observe high rates of secondary deletions at duplicated sites, with 8% of duplicated sites in D. simulans and 17% of sites in D. yakuba modified with deletions. These secondary deletions are consistent with the action of the large loop mismatch repair system acting to remove polymorphic tandem duplication, resulting in rapid dynamics of gain and loss in duplicated alleles and a richer substrate of genetic novelty than has been previously reported. Most duplications are present in only single strains, suggesting deleterious impacts are common. D. simulans shows larger numbers of whole gene duplications in comparison to larger proportions of gene fragments in D. yakuba. D. simulans displays an excess of high frequency variants on the X chromosome, consistent with adaptive evolution through duplications on the D. simulans X or demographic forces driving duplicates to high frequency. We identify 78 chimeric genes in D. yakuba and 38 chimeric genes in D. simulans, as well as 143 cases of recruited non-coding sequence in D. yakuba and 96 in D. simulans, in agreement with rates of chimeric gene origination in D. melanogaster. Together, these results suggest that tandem duplications often result in complex variation beyond whole gene duplications that offers a rich substrate of standing variation that is likely to contribute both to detrimental phenotypes and disease, as well as to adaptive evolutionary change.Comment: Revised Version- Accepted at Molecular Biology and Evolutio