scholarly journals False negatives in GBA1 sequencing due to polymerase dependent allelic imbalance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jonas M. den Heijer ◽  
Arnoud Schmitz ◽  
Peter Lansbury ◽  
Valerie C. Cullen ◽  
Dana C. Hilt ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Allele Frequency ◽  
Allelic Imbalance ◽  
Annealing Temperature ◽  
Variant Calling ◽  
Next Generation ◽  
Negative Results ◽  
Exonic Variant ◽  
Primer Annealing ◽  
Generation Sequencing

AbstractA variant in the GBA1 gene is one of the most common genetic risk factors to develop Parkinson’s disease (PD). Here the serendipitous finding is reported of a polymerase dependent allelic imbalance when using next generation sequencing, potentially resulting in false-negative results when the allele frequency falls below the variant calling threshold (by default commonly at 30%). The full GBA1 gene was sequenced using next generation sequencing on saliva derived DNA from PD patients. Four polymerase chain reaction conditions were varied in twelve samples, to investigate the effect on allelic imbalance: (1) the primers (n = 4); (2) the polymerase enzymes (n = 2); (3) the primer annealing temperature (Ta) specified for the used polymerase; and (4) the amount of DNA input. Initially, 1295 samples were sequenced using Q5 High-Fidelity DNA Polymerase. 112 samples (8.6%) had an exonic variant and an additional 104 samples (8.0%) had an exonic variant that did not pass the variant frequency calling threshold of 30%. After changing the polymerase to TaKaRa LA Taq DNA Polymerase Hot-Start Version: RR042B, all samples had an allele frequency passing the calling threshold. Allele frequency was unaffected by a change in primer, annealing temperature or amount of DNA input. Sequencing of the GBA1 gene using next generation sequencing might be susceptible to a polymerase specific allelic imbalance, which can result in a large amount of flase-negative results. This was resolved in our case by changing the polymerase. Regions displaying low variant calling frequencies in GBA1 sequencing output in previous and future studies might warrant additional scrutiny.

High throughput crop genome genotyping by a combination of pool next generation sequencing and haplotype-based data processing

2021 ◽  
Author(s):  
Michael Schneider ◽  
Asis Shrestha ◽  
Agim Ballvora ◽  
Jens Leon
Keyword(s):  
Next Generation Sequencing ◽  
Allele Frequency ◽  
Frequency Estimation ◽  
Whole Genome ◽  
Next Generation ◽  
Conservation Genomics ◽  
High Coverage ◽  
Allele Frequency Estimation ◽  
Low Coverage ◽  
Generation Sequencing

Abstract BackgroundThe identification of environmentally specific alleles and the observation of evolutional processes is a goal of conservation genomics. By generational changes of allele frequencies in populations, questions regarding effective population size, gene flow, drift, and selection can be addressed. The observation of such effects often is a trade-off of costs and resolution, when a decent sample of genotypes should be genotyped for many loci. Pool genotyping approaches can derive a high resolution and precision in allele frequency estimation, when high coverage sequencing is utilized. Still, pool high coverage pool sequencing of big genomes comes along with high costs.ResultsHere we present a reliable method to estimate a barley population’s allele frequency at low coverage sequencing. Three hundred genotypes were sampled from a barley backcross population to estimate the entire population’s allele frequency. The allele frequency estimation accuracy and yield were compared for three next generation sequencing methods. To reveal accurate allele frequency estimates on a low coverage sequencing level, a haplotyping approach was performed. Low coverage allele frequency of positional connected single polymorphisms were aggregated to a single haplotype allele frequency, resulting in two to 271 times higher depth and increased precision. We compared different haplotyping tactics, showing that gene and chip marker-based haplotypes perform on par or better than simple contig haplotype windows. The comparison of multiple pool samples and the referencing against an individual sequencing approach revealed whole genome pool resequencing having the highest correlation to individual genotyping (up to 0.97), while transcriptomics and genotyping by sequencing indicated higher error rates and lower correlations.ConclusionUsing the proposed method allows to identify the allele frequency of populations with high accuracy at low cost. This is particularly interesting for conservation genomics in species with big genomes, like barley or wheat. Whole genome low coverage resequencing at 10x coverage can deliver a highly accurate estimation of the allele frequency, when a loci-based haplotyping approach is applied. Using annotated haplotypes allows to capitalize from biological background and statistical robustness.

scholarly journals Improved next-generation sequencing pre-capture library yields and sequencing parameters using on-bead PCR

BioTechniques ◽  
2020 ◽  
Vol 68 (1) ◽  
pp. 48-51 ◽  
Author(s):  
Christopher R McEvoy ◽  
Timothy Semple ◽  
Bhargavi Yellapu ◽  
David Y Choong ◽  
Huiling Xu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Limiting Factor ◽  
Next Generation ◽  
Tumor Biopsy ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Next Generation Sequencing Ngs ◽  
Tumor Dna ◽  
Generation Sequencing

Tumor DNA sequencing results can have important clinical implications. However, its use is often limited by low DNA input, owing to small tumor biopsy size. To help overcome this limitation we have developed a simple improvement to a commonly used next-generation sequencing (NGS) capture-based library preparation method using formalin-fixed paraffin-embedded-derived tumor DNA. By using on-bead PCR for pre-capture library generation we show that library yields are dramatically increased, resulting in decreased sample failure rates. Improved yields allowed for a reduction in PCR cycles, which translated into improved sequencing parameters without affecting variant calling. This methodology should be applicable to any NGS system in which input DNA is a limiting factor.

scholarly journals A customized scaffolds approach for the detection and phasing of complex variants by next-generation sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiandong Zeng ◽  
Natalia T. Leach ◽  
Zhaoqing Zhou ◽  
Hui Zhu ◽  
Jean A. Smith ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Causative Mutation ◽  
Next Generation ◽  
Single Nucleotide ◽  
Highly Sensitive ◽  
Highly Sensitive Detection ◽  
Next Generation Sequencing Ngs ◽  
Reference Bias ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) is widely used in genetic testing for the highly sensitive detection of single nucleotide changes and small insertions or deletions. However, detection and phasing of structural variants, especially in repetitive or homologous regions, can be problematic due to uneven read coverage or genome reference bias, resulting in false calls. To circumvent this challenge, a computational approach utilizing customized scaffolds as supplementary reference sequences for read alignment was developed, and its effectiveness demonstrated with two CBS gene variants: NM_000071.2:c.833T>C and NM_000071.2:c.[833T>C; 844_845ins68]. Variant c.833T>C is a known causative mutation for homocystinuria, but is not pathogenic when in cis with the insertion, c.844_845ins68, because of alternative splicing. Using simulated reads, the custom scaffolds method resolved all possible combinations with 100% accuracy and, based on > 60,000 clinical specimens, exceeded the performance of current approaches that only align reads to GRCh37/hg19 for the detection of c.833T>C alone or in cis with c.844_845ins68. Furthermore, analysis of two 1000 Genomes Project trios revealed that the c.[833T>C; 844_845ins68] complex variant had previously been undetected in these datasets, likely due to the alignment method used. This approach can be configured for existing workflows to detect other challenging and potentially underrepresented variants, thereby augmenting accurate variant calling in clinical NGS testing.

scholarly journals Validation and assessment of variant calling pipelines for next-generation sequencing

2014 ◽  
Vol 8 (1) ◽  
pp. 14 ◽  
Author(s):  
Mehdi Pirooznia ◽  
Melissa Kramer ◽  
Jennifer Parla ◽  
Fernando S Goes ◽  
James B Potash ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Next Generation ◽  
Generation Sequencing

scholarly journals Lacer: accurate base quality score recalibration for improving variant calling from next-generation sequencing data in any organism

2017 ◽  
Author(s):  
Jade C.S. Chung ◽  
Swaine L. Chen
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Quality Score ◽  
Identification Accuracy ◽  
Next Generation Sequencing Data ◽  
Sequencing Error ◽  
Next Generation ◽  
Sequencing Data ◽  
Base Quality Score ◽  
Generation Sequencing

AbstractNext-generation sequencing data is accompanied by quality scores that quantify sequencing error. Inaccuracies in these quality scores propagate through all subsequent analyses; thus base quality score recalibration is a standard step in many next-generation sequencing workflows, resulting in improved variant calls. Current base quality score recalibration algorithms rely on the assumption that sequencing errors are already known; for human resequencing data, relatively complete variant databases facilitate this. However, because existing databases are still incomplete, recalibration is still inaccurate; and most organisms do not have variant databases, exacerbating inaccuracy for non-human data. To overcome these logical and practical problems, we introduce Lacer, which recalibrates base quality scores without assuming knowledge of correct and incorrect bases and without requiring knowledge of common variants. Lacer is the first logically sound, fully general, and truly accurate base recalibrator. Lacer enhances variant identification accuracy for resequencing data of human as well as other organisms (which are not accessible to current recalibrators), simultaneously improving and extending the benefits of base quality score recalibration to nearly all ongoing sequencing projects. Lacer is available at: https://github.com/swainechen/lacer.

scholarly journals Case Report: A Severe and Multi-Site Nocardia farcinica Infection Rapidly and Precisely Identified by Metagenomic Next-Generation Sequencing

2021 ◽  
Vol 8 ◽  
Author(s):  
Mengfan Jiao ◽  
Xiang Deng ◽  
Hongfu Yang ◽  
Junqiang Dong ◽  
Jun Lv ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Venous Blood ◽  
Opportunistic Pathogen ◽  
Diagnosis And Treatment ◽  
Next Generation ◽  
Nocardia Farcinica ◽  
Negative Results ◽  
Golden Standard ◽  
Immunosuppressed Patients ◽  
Generation Sequencing

Nocardia genus is an aerobic, gram-positive, and opportunistic pathogen, which mainly affects cell-mediated immunosuppressed patients. Early diagnosis and treatment greatly improve prognosis. However, the limitation of golden standard-bacterial culture exists. Here, we report a 61-year-old male with pneumonia, sepsis and intermuscular abscesses induced by Nocardia farcinica. Venous blood culture reported negative results. Former improper diagnosis and treatment did not improve his condition. With the assistant of metagenomic next-generation sequencing, the pathogen was identified as Nocardia farcinica. He was then applied with accurate treatment and had a remarkable clinical and radiological improvement.

scholarly journals Pisces: An Accurate and Versatile Variant Caller for Somatic and Germline Next-Generation Sequencing Data

2018 ◽  
Author(s):  
Tamsen Dunn ◽  
Gwenn Berry ◽  
Dorothea Emig-Agius ◽  
Yu Jiang ◽  
Serena Lei ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Gene Mutations ◽  
Variant Calling ◽  
Amplicon Sequencing ◽  
Supplementary Information ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Ras Gene ◽  
Generation Sequencing

AbstractMotivationNext-Generation Sequencing (NGS) technology is transitioning quickly from research labs to clinical settings. The diagnosis and treatment selection for many acquired and autosomal conditions necessitate a method for accurately detecting somatic and germline variants, suitable for the clinic.ResultsWe have developed Pisces, a rapid, versatile and accurate small variant calling suite designed for somatic and germline amplicon sequencing applications. Pisces accuracy is achieved by four distinct modules, the Pisces Read Stitcher, Pisces Variant Caller, the Pisces Variant Quality Recalibrator, and the Pisces Variant Phaser. Each module incorporates a number of novel algorithmic strategies aimed at reducing noise or increasing the likelihood of detecting a true variant.AvailabilityPisces is distributed under an open source license and can be downloaded from https://github.com/Illumina/Pisces. Pisces is available on the BaseSpace™ SequenceHub as part of the TruSeq Amplicon workflow and the Illumina Ampliseq Workflow. Pisces is distributed on Illumina sequencing platforms such as the MiSeq™, and is included in the Praxis™ Extended RAS Panel test which was recently approved by the FDA for the detection of multiple RAS gene [email protected] informationSupplementary data are available online.

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