A Practical Guide To Filtering and Prioritizing Genetic Variants

Dashti, Mahjoubeh Jalali Sefid; Gamieldien, Junaid

doi:10.2144/000114492

Cited by 65 publications

(44 citation statements)

References 75 publications

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“…Deviation from HWE may occur more frequently in specific genetic regions (e.g., near centromeres 96 ), and SNPs in these could be excluded from the target pool. Numerous other filtering options – e.g., based on allele count (to enhance resolution per SNP), distance to insertion-deletions (to improve target alignment), or percent missing information (to avoid poorly mapping regions) – are easily implemented with common analysis tools 97 .…”

Section: Discussionmentioning

confidence: 99%

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

Schwabl¹,

et al. 2020

Preprint

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20Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful 21 inference from pathogen genetic variation, however, is often restrained by limited access to representative 22 target DNA, especially in the study of obligate parasitic species for which ex vivo culture is resource-intensive 23 or bias-prone. Modern sequence capture methods enable pathogen genetic variation to be analyzed directly 24 from vector/host material but are often too complex and expensive for resource-poor settings where infectious 25 diseases prevail. This study proposes a simple, cost-effective 'genome-wide locus sequence typing' (GLST) 26 tool based on massive parallel amplification of information hotspots throughout the target pathogen genome. 27 The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a 28 single reaction tube, and subsequent agarose gel-based clean-up and barcoding completes library preparation 29 at under 4 USD per sample. Approximately 100 libraries can be sequenced together in one Illumina MiSeq 30 run. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic 31 agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free 32 metagenomic extracts from triatomine vectors containing a minimum of 3.69 pg/µl T. cruzi DNA and further 33 elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing 34 discrete typing units (DTUs) TcI, TcIII, TcIV, and TcVI. The 780 SNP sites we identify in the sample set 35 repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and 36 geographic distances at regional (< 150 km) as well as continental scales. The markers also clearly separate 37 DTUs. We discuss the advantages, limitations and prospects of our method across a spectrum of 38 epidemiological research. 40Genome-wide single nucleotide polymorphism (SNP) analysis is a powerful and increasingly common 41 approach in the study and surveillance of infectious disease. Understanding patterns of SNP diversity within 42 pathogen genomes and across pathogen populations can resolve fundamental biological questions (e.g., 43 reproductive mechanisms in T. cruzi 1 , reconstruct past 2 and present transmission networks (e.g., 44 Staphylococcus infections within hospitals) 3 or identify the genetic bases of virulence 4,5 and resistance to drugs 45 (see examples from Plasmodium spp. 6,7 ). A number of obstacles, however, complicate access to 46 representative, genome-wide SNP information using modern sequencing tools. Micro-pathogens are often 47 sampled in low quantities and together with large amounts of host/vector tissue, microbiota, or environmental 48 DNA. Sequencing is rarely viable directly from the infection source and studies have often found it necessary 49 to isolate and culture the target organism to higher densities before extracting D...

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Section: Discussionmentioning

confidence: 99%

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

Schwabl¹,

et al. 2020

Preprint

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“…With the aim of establishing the genetic variants that may act as drivers of the disease, the VarSEQ™ software (Golden Helix) was used. Variant Call Format (VCF) files were filtered according to strict criteria: a reading depth of at least 15x, a gnomAD global frequency <1%, high impact (frameshift, splice variants, stop-loss and stop-gain variants), and a detectable presence in at least 15% of the reads 20 .…”

Section: Methodsmentioning

confidence: 99%

Comprehensive characterization of the mutational landscape in localized anal squamous cell carcinoma

Trilla-Fuertes¹,

Ghanem

Maurel

et al. 2019

Preprint

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PurposeAnal squamous cell carcinoma (ASCC) is a rare neoplasm. Chemo-radiotherapy is the standard of care, with no therapeutic advances achieved over the past three decades. Thus, a deeper molecular characterization of this disease is still necessary.MethodsWe analyzed 46 paraffin-embedded tumor samples from patients diagnosed with primary ASCC by exome sequencing. A bioinformatics approach focused in the identification of high impact genetic variants, which may act as drivers of oncogenesis, was performed. The relation between genetics variant and prognosis was also studied.ResultsThe list of high impact genetic variants was unique for each patient. However, the pathways in which these genes are involved are well-known hallmarks of cancer, such as angiogenesis or immune pathways. Additionally, we determined that genetic variants in BRCA2, ZNF750, FAM208B, ZNF599 and ZC3H13 genes are related with poor disease-free survival in ASCC.ConclusionHigh impact genetic variants in ASCC affect pathways involved in cancer development, and may play a role in the etiology of the disease. Variants in BRCA2, ZNF750, FAM208B, ZNF599 and ZC3H13 genes seem to be related with prognosis in ASCC. Sequencing of ASCC clinical samples appears an encouraging tool for the molecular portrait of this disease.

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“…The subsequent interpretation of variants is an even greater challenge. 45 Thus, several filters can already significantly reduce the number of candidate variants (►Supplemental Material) (►Fig. 2).…”

Section: Targeted Gene Sequencingmentioning

confidence: 99%

Molecular Diagnosis of Inherited Coagulation and Bleeding Disorders

Bastida

Benito

Lozano

et al. 2019

Semin Thromb Hemost

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Diagnosis of inherited bleeding disorders (IBDs) remains challenging, especially in the case of inherited platelet disorders, due to the heterogeneity of the clinical and laboratory phenotype, the limited specificity of platelet function tests, and the large number of potential culprit genes. Unraveling the underlying molecular defect provides the definitive diagnosis of IBDs, facilitating prognosis and clinical care, which are especially important for severe clinical syndromes and those that may be associated with an increased risk of malignancy. Until recently, Sanger sequencing of candidate genes has been the only method of molecular diagnosis, but this approach is time-consuming and costly and requires phenotype-based identification of any obvious candidate gene(s). Nowadays, high-throughput sequencing (HTS) allows the simultaneous and rapid investigation of multiple genes at a manageable cost. This HTS technology that includes targeted sequencing of prespecified genes, whole-exome sequencing, or whole-genome sequencing, is revolutionizing the genetic diagnosis of human diseases. Through its extensive implementation in research and clinical practice, HTS is rapidly improving the molecular characterization of IBDs. However, despite the availability of this powerful approach, many patients still do not receive a diagnosis. As IBDs are complex and rare diseases, development of more advanced laboratory assays, improvements in bioinformatic pipelines, and the formation of multidisciplinary teams are encouraged to advance our understanding of IBDs.

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A Practical Guide To Filtering and Prioritizing Genetic Variants

Cited by 65 publications

References 75 publications

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

Genome-wide locus sequence typing (GLST) of eukaryotic pathogens

Comprehensive characterization of the mutational landscape in localized anal squamous cell carcinoma

Molecular Diagnosis of Inherited Coagulation and Bleeding Disorders

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