scholarly journals Longshot: accurate variant calling in diploid genomes using single-molecule long read sequencing

2019 ◽  
Author(s):  
Peter Edge ◽  
Vikas Bansal
Keyword(s):  
Single Molecule ◽  
Variant Calling ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Short Reads ◽  
Pacific Biosciences ◽  
Sequencing Technologies ◽  
Accurate Detection ◽  
Oxford Nanopore

AbstractShort-read sequencing technologies such as Illumina enable the accurate detection of single nucleotide variants (SNVs) and short insertion/deletion variants in human genomes but are unable to provide information about haplotypes and variants in repetitive regions of the genome. Single-molecule sequencing technologies such as Pacific Biosciences and Oxford Nanopore generate long reads (≥ 10 kb in length) that can potentially address these limitations of short reads. However, the high error rate of SMS reads makes it challenging to detect small-scale variants in diploid genomes. We introduce a variant calling method, Longshot, that leverages the haplotype information present in SMS reads to enable the accurate detection and phasing of single nucleotide variants in diploid genomes. Using whole-genome Pacific Biosciences data for multiple human individuals, we demonstrate that Longshot achieves very high accuracy for SNV detection (precision ≥0.992 and recall ≥0.96) that is significantly better than existing variant calling methods. Longshot can also call SNVs with good accuracy using whole-genome Oxford Nanopore data. Finally, we demonstrate that it enables the discovery of variants in duplicated regions of the genome that cannot be mapped using short reads. Longshot is freely available at https://github.com/pjedge/longshot.

scholarly journals Longshot enables accurate variant calling in diploid genomes from single-molecule long read sequencing

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Peter Edge ◽  
Vikas Bansal
Keyword(s):  
Single Molecule ◽  
Variant Calling ◽  
Small Scale ◽  
Whole Genome ◽  
Limited Information ◽  
Single Nucleotide Variants ◽  
Pacific Biosciences ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read

Abstract Whole-genome sequencing using sequencing technologies such as Illumina enables the accurate detection of small-scale variants but provides limited information about haplotypes and variants in repetitive regions of the human genome. Single-molecule sequencing (SMS) technologies such as Pacific Biosciences and Oxford Nanopore generate long reads that can potentially address the limitations of short-read sequencing. However, the high error rate of SMS reads makes it challenging to detect small-scale variants in diploid genomes. We introduce a variant calling method, Longshot, which leverages the haplotype information present in SMS reads to accurately detect and phase single-nucleotide variants (SNVs) in diploid genomes. We demonstrate that Longshot achieves very high accuracy for SNV detection using whole-genome Pacific Biosciences data, outperforms existing variant calling methods, and enables variant detection in duplicated regions of the genome that cannot be mapped using short reads.

scholarly journals Accurate detection of subclonal single nucleotide variants in whole genome amplified and pooled cancer samples using HaloPlex target enrichment

BMC Genomics ◽  
2013 ◽  
Vol 14 (1) ◽  
pp. 856 ◽  
Author(s):  
Eva C Berglund ◽  
Carl Lindqvist ◽  
Shahina Hayat ◽  
Elin Övernäs ◽  
Niklas Henriksson ◽  
...  
Keyword(s):  
Whole Genome ◽  
Target Enrichment ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Accurate Detection

scholarly journals Comparison of single-nucleotide variants identified by Illumina and Oxford Nanopore technologies in the context of a potential outbreak of Shiga toxin–producing Escherichia coli

GigaScience ◽  
2019 ◽  
Vol 8 (8) ◽  
Author(s):  
David R Greig ◽  
Claire Jenkins ◽  
Saheer Gharbia ◽  
Timothy J Dallman
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Variant Calling ◽  
Reference Database ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Oxford Nanopore ◽  
Variant Filtering ◽  
Oxford Nanopore Technologies

Abstract Background We aimed to compare Illumina and Oxford Nanopore Technology sequencing data from the 2 isolates of Shiga toxin–producing Escherichia coli (STEC) O157:H7 to determine whether concordant single-nucleotide variants were identified and whether inference of relatedness was consistent with the 2 technologies. Results For the Illumina workflow, the time from DNA extraction to availability of results was ∼40 hours, whereas with the ONT workflow serotyping and Shiga toxin subtyping variant identification were available within 7 hours. After optimization of the ONT variant filtering, on average 95% of the discrepant positions between the technologies were accounted for by methylated positions found in the described 5-methylcytosine motif sequences, CC(A/T)GG. Of the few discrepant variants (6 and 7 difference for the 2 isolates) identified by the 2 technologies, it is likely that both methodologies contain false calls. Conclusions Despite these discrepancies, Illumina and Oxford Nanopore Technology sequences from the same case were placed on the same phylogenetic location against a dense reference database of STEC O157:H7 genomes sequenced using the Illumina workflow. Robust single-nucleotide polymorphism typing using MinION-based variant calling is possible, and we provide evidence that the 2 technologies can be used interchangeably to type STEC O157:H7 in a public health setting.

scholarly journals Detecting and phasing minor single-nucleotide variants from long-read sequencing data

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhixing Feng ◽  
Jose C. Clemente ◽  
Brandon Wong ◽  
Eric E. Schadt
Keyword(s):  
Genetic Heterogeneity ◽  
Error Rates ◽  
Metagenomic Data ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Technological Limitations

AbstractCellular genetic heterogeneity is common in many biological conditions including cancer, microbiome, and co-infection of multiple pathogens. Detecting and phasing minor variants play an instrumental role in deciphering cellular genetic heterogeneity, but they are still difficult tasks because of technological limitations. Recently, long-read sequencing technologies, including those by Pacific Biosciences and Oxford Nanopore, provide an opportunity to tackle these challenges. However, high error rates make it difficult to take full advantage of these technologies. To fill this gap, we introduce iGDA, an open-source tool that can accurately detect and phase minor single-nucleotide variants (SNVs), whose frequencies are as low as 0.2%, from raw long-read sequencing data. We also demonstrate that iGDA can accurately reconstruct haplotypes in closely related strains of the same species (divergence ≥0.011%) from long-read metagenomic data.

scholarly journals Hapo-G, Haplotype-Aware Polishing Of Genome Assemblies

2020 ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
Short Reads ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

Single-molecule sequencing technologies have recently been commercialized by Pacific Biosciences and Oxford Nanopore with the promise of sequencing long DNA fragments (kilobases to megabases order) and then, using efficient algorithms, provide high quality assemblies in terms of contiguity and completeness of repetitive regions. However, the error rate of long-read technologies is higher than that of short-read technologies. This has a direct consequence on the base quality of genome assemblies, particularly in coding regions where sequencing errors can disrupt the coding frame of genes. In the case of diploid genomes, the consensus of a given gene can be a mixture between the two haplotypes and can lead to premature stop codons. Several methods have been developed to polish genome assemblies using short reads and generally, they inspect the nucleotide one by one, and provide a correction for each nucleotide of the input assembly. As a result, these algorithms are not able to properly process diploid genomes and they typically switch from one haplotype to another. Herein we proposed Hapo-G (Haplotype-Aware Polishing Of Genomes), a new algorithm capable of incorporating phasing information from short reads to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

scholarly journals Detecting and phasing minor single-nucleotide variants from long-read sequencing data

2020 ◽  
Author(s):  
Zhixing Feng ◽  
Jose Clemente ◽  
Brandon Wong ◽  
Eric E. Schadt
Keyword(s):  
Genetic Heterogeneity ◽  
Error Rates ◽  
Metagenomic Data ◽  
Significant Advance ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read

AbstractCellular genetic heterogeneity is common in many biological conditions including cancer, microbiome, co-infection of multiple pathogens. Detecting and phasing minor variants, which is to determine whether multiple variants are from the same haplotype, play an instrumental role in deciphering cellular genetic heterogeneity, but are still difficult because of technological limitations. Recently, long-read sequencing technologies, including those by Pacific Biosciences and Oxford Nanopore, have provided an unprecedented opportunity to tackle these challenges. However, high error rates make it difficult to take full advantage of these technologies. To fill this gap, we introduce iGDA, an open-source tool that can accurately detect and phase minor single-nucleotide variants (SNVs), whose frequencies are as low as 0.2%, from raw long-read sequencing data. We also demonstrated that iGDA can accurately reconstruct haplotypes in closely-related strains of the same species (divergence ≥ 0.011%) from long-read metagenomic data. Our approach, therefore, presents a significant advance towards the complete deciphering of cellular genetic heterogeneity.

scholarly journals Comparison of Multiple Displacement Amplification (MDA) and Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) in Limited DNA Sequencing Based on Tube and Droplet

Micromachines ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 645
Author(s):  
Xiaoxiang Zhou ◽  
Ying Xu ◽  
Libo Zhu ◽  
Zhen Su ◽  
Xiaoming Han ◽  
...  
Keyword(s):  
Single Molecule ◽  
Multiple Displacement Amplification ◽  
Genomic Alteration ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Amplification Bias ◽  
Advantages And Disadvantages ◽  
Droplet Method

Whole genome amplification (WGA) is crucial for whole genome sequencing to investigate complex genomic alteration at the single-cell or even single-molecule level. Multiple displacement amplification (MDA) and multiple annealing and looping based amplification cycles (MALBAC) are two most widely applied WGA methods, which have different advantages and disadvantages, dependent on research objectives. Herein, we compared the MDA and MALBAC to provide more information on their performance in droplets and tubes. We observed that the droplet method could dramatically reduce the amplification bias and retain the high accuracy of replication than the conventional tube method. Furthermore, the droplet method exhibited higher efficiency and sensitivity for both homozygous and heterozygous single nucleotide variants (SNVs) at the low sequencing depth. In addition, we also found that MALBAC offered a greater uniformity and reproducibility and MDA showed a better efficiency of genomic coverage and SNV detection. Our results provided insights that will allow future decision making.

scholarly journals Comparison of single nucleotide variants identified by Illumina and Oxford Nanopore technologies in the context of a potential outbreak of Shiga Toxin ProducingEscherichia coli

2019 ◽  
Author(s):  
David R Greig ◽  
Claire Jenkins ◽  
Saheer Gharbia ◽  
Timothy J Dallman
Keyword(s):  
Shiga Toxin ◽  
Variant Calling ◽  
Reference Database ◽  
Sequencing Data ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Oxford Nanopore ◽  
Variant Filtering ◽  
Oxford Nanopore Technologies ◽  
Variant Identification

AbstractBackgroundWe aimed to compare Illumina and Oxford Nanopore Technology (ONT) sequencing data from the two isolates of STEC O157:H7 to determine whether concordant single nucleotide variants were identified and whether inference of relatedness was consistent with the two technologies.ResultsFor the Illumina workflow, the time from DNA extraction to availability of results, was approximately 40 hours in comparison to the ONT workflow where serotyping, Shiga toxin subtyping variant identification were available within seven hours. After optimisation of the ONT variant filtering, on average 95% of the discrepant positions between the technologies were accounted for by methylated positions found in the described 5-Methylcytosine motif sequences, CC(A/T)GG. Of the few discrepant variants (6 and 7 difference for the two isolates) identified by the two technologies, it is likely that both methodologies contain false calls.ConclusionsDespite these discrepancies, Illumina and ONT sequences from the same case were placed on the same phylogenetic location against a dense reference database of STEC O157:H7 genomes sequenced using the Illumina workflow. Robust SNP typing using MinION-based variant calling is possible and we provide evidence that the two technologies can be used interchangeably to type STEC O157:H7 in a public health setting.

scholarly journals Unsuspected somatic mosaicism for FBN1 gene contributes to Marfan syndrome

2021 ◽  
Author(s):  
Pauline Arnaud ◽  
Hélène Morel ◽  
Olivier Milleron ◽  
Laurent Gouya ◽  
Christine Francannet ◽  
...  
Keyword(s):  
Marfan Syndrome ◽  
Somatic Mosaicism ◽  
Variant Calling ◽  
Copy Number Variations ◽  
Pathogenic Variant ◽  
Single Nucleotide Variants ◽  
Bioinformatics Analyses ◽  
Single Nucleotide ◽  
Fbn1 Gene ◽  
Pathogenic Variants

Abstract Purpose Individuals with mosaic pathogenic variants in the FBN1 gene are mainly described in the course of familial screening. In the literature, almost all these mosaic individuals are asymptomatic. In this study, we report the experience of our team on more than 5,000 Marfan syndrome (MFS) probands. Methods Next-generation sequencing (NGS) capture technology allowed us to identify five cases of MFS probands who harbored a mosaic pathogenic variant in the FBN1 gene. Results These five sporadic mosaic probands displayed classical features usually seen in Marfan syndrome. Combined with the results of the literature, these rare findings concerned both single-nucleotide variants and copy-number variations. Conclusion This underestimated finding should not be overlooked in the molecular diagnosis of MFS patients and warrants an adaptation of the parameters used in bioinformatics analyses. The five present cases of symptomatic MFS probands harboring a mosaic FBN1 pathogenic variant reinforce the fact that apparently asymptomatic mosaic parents should have a complete clinical examination and a regular cardiovascular follow-up. We advise that individuals with a typical MFS for whom no single-nucleotide pathogenic variant or exon deletion/duplication was identified should be tested by NGS capture panel with an adapted variant calling analysis.

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