scholarly journals A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Sophie Dhorne-Pollet ◽  
Eric Barrey ◽  
Nicolas Pollet
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Dna ◽  
Multiple Displacement Amplification ◽  
Variant Calling ◽  
Ground Truth ◽  
Mitochondrial Genomes ◽  
Long Reads ◽  
Long Read ◽  
Variant Analysis ◽  
Mitochondrial Dna Variants

Abstract Background Mitochondrial DNA is remarkably polymorphic. This is why animal geneticists survey mitochondrial genomes variations for fundamental and applied purposes. We present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step. Results We optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more of the whole mtDNA provided a coverage that varied between 118X and 488X. We evaluated SNPs identified using these long-reads by Sanger sequencing as ground truth and found a precision of 100.0%; a recall of 93.1% and a F1-score of 0.964 using the Twilight horse mtDNA reference. The choice of the mtDNA reference impacted variant calling efficiency with F1-scores varying between 0.947 and 0.964. Conclusions Our method to amplify mtDNA and to sequence it using the nanopore technology is usable for mitochondrial DNA variant analysis. With minor modifications, this approach could easily be applied to other large circular DNA molecules.

scholarly journals A new method for long-read sequencing of animal mitochondrial genomes: application to the identification of equine mitochondrial DNA variants

2019 ◽  
Author(s):  
Sophie Dhorne-Pollet ◽  
Eric Barrey ◽  
Nicolas Pollet
Keyword(s):  
Mitochondrial Dna ◽  
Nuclear Dna ◽  
Multiple Displacement Amplification ◽  
Mitochondrial Genomes ◽  
Sequencing Technology ◽  
Long Reads ◽  
Selective Elimination ◽  
Long Read ◽  
Variant Analysis ◽  
Mitochondrial Dna Variants

AbstractBackgroundWe present here an approach to sequence whole mitochondrial genomes using nanopore long-read sequencing. Our method relies on the selective elimination of nuclear DNA using an exonuclease treatment and on the amplification of circular mitochondrial DNA using a multiple displacement amplification step.ResultsWe optimized each preparative step to obtain a 100 million-fold enrichment of horse mitochondrial DNA relative to nuclear DNA. We sequenced these amplified mitochondrial DNA using nanopore sequencing technology and obtained mitochondrial DNA reads that represented up to half of the sequencing output. The sequence reads were 2.3 kb of mean length and provided an even coverage of the mitochondrial genome. Long-reads spanning half or more of the whole mtDNA provided a coverage that varied between 118X and 488X. Finally, we identified SNPs with a precision of 98.1%; recall of 85.2% and a F1-score of 0.912.ConclusionsOur analyses show that our method to amplify mtDNA and to sequence it using the nanopore technology is usable for mitochondrial DNA variant analysis. With minor modifications, this approach could easily be applied to other large circular DNA molecules.

scholarly journals The application of Nanopore sequencing for variant calling on the human mitochondrial DNA

2021 ◽  
Vol 66 (2) ◽  
Author(s):  
Anton Shikov ◽  
Viktoriya Tsay ◽  
Mikhail Fedyakov ◽  
Yuri Eismont ◽  
Alena Rudnik ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
False Negative ◽  
Variant Calling ◽  
Illumina Miseq ◽  
Routine Practice ◽  
Jaccard Coefficient ◽  
High Coverage ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Long Read

The emergence of long-read sequencing technologies has made a revolutionary step in genome biology and medicine. However, long reads are characterized by a relatively high error rate, impairing their usage for variant calling as a part of routine practice. Thus, we here examine different popular variant callers on long-read sequences of the human mitochondrial genome, convenient in terms of small size and easily obtained high coverage. The sequencing of mitochondrial DNA from 8 patients was conducted via Illumina (MiSeq) and the Oxford Nanopore platform (MinION), with the former utilized as a gold standard when evaluating variant calling’s accuracy. We used a conventional GATK3-BWA-based pipeline for paired-end reads and Guppy basecaller coupled with minimap2 for MinION data, respectively. We then compared the outputs of Clairvoyante, Nanopolish, GATK3, Longshot, DeepVariant, and Varscan tools applied on long-read alignments by analyzing false-positive and false-negative rates. While for most callers, raw signals represented false positives due to homopolymeric errors, Nanopolish demonstrated both high similarity (Jaccard coefficient of 0.82) and a comparable number of calls with the Illumina data (140 vs. 154) with the best performance according to AUC (area under ROC curve, 0.953) as well. In sum, our results, despite being obtained from a small dataset, provide evidence that sufficient coverage coupled with an optimal pipeline could make long reads of mitochondrial DNA applicable for variant calling.

scholarly journals Fast and sensitive mapping of error-prone nanopore sequencing reads with GraphMap

2015 ◽  
Author(s):  
Ivan Sovic ◽  
Mile Sikic ◽  
Andreas Wilm ◽  
Shannon Nicole Fenlon ◽  
Swaine Chen ◽  
...  
Keyword(s):  
Human Genome ◽  
Variant Calling ◽  
Error Rates ◽  
Nanopore Sequencing ◽  
Structural Variants ◽  
Specific Identification ◽  
Long Reads ◽  
Long Read ◽  
Specific Error ◽  
Very High

Exploiting the power of nanopore sequencing requires the development of new bioinformatics approaches to deal with its specific error characteristics. We present the first nanopore read mapper (GraphMap) that uses a read-funneling paradigm to robustly handle variable error rates and fast graph traversal to align long reads with speed and very high precision (>95%). Evaluation on MinION sequencing datasets against short and long-read mappers indicates that GraphMap increases mapping sensitivity by at least 15-80%. GraphMap alignments are the first to demonstrate consensus calling with <1 error in 100,000 bases, variant calling on the human genome with 76% improvement in sensitivity over the next best mapper (BWA-MEM), precise detection of structural variants from 100bp to 4kbp in length and species and strain-specific identification of pathogens using MinION reads. GraphMap is available open source under the MIT license at https://github.com/isovic/graphmap.

scholarly journals Evaluation of Germline Structural Variant Calling Methods for Nanopore Sequencing Data

2021 ◽  
Vol 12 ◽  
Author(s):  
Davide Bolognini ◽  
Alberto Magi
Keyword(s):  
Variant Calling ◽  
Research Report ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Factors Affecting ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Sequencing Studies ◽  
Long Read

Structural variants (SVs) are genomic rearrangements that involve at least 50 nucleotides and are known to have a serious impact on human health. While prior short-read sequencing technologies have often proved inadequate for a comprehensive assessment of structural variation, more recent long reads from Oxford Nanopore Technologies have already been proven invaluable for the discovery of large SVs and hold the potential to facilitate the resolution of the full SV spectrum. With many long-read sequencing studies to follow, it is crucial to assess factors affecting current SV calling pipelines for nanopore sequencing data. In this brief research report, we evaluate and compare the performances of five long-read SV callers across four long-read aligners using both real and synthetic nanopore datasets. In particular, we focus on the effects of read alignment, sequencing coverage, and variant allele depth on the detection and genotyping of SVs of different types and size ranges and provide insights into precision and recall of SV callsets generated by integrating the various long-read aligners and SV callers. The computational pipeline we propose is publicly available at https://github.com/davidebolo1993/EViNCe and can be adjusted to further evaluate future nanopore sequencing datasets.

scholarly journals NanoCaller for accurate detection of SNPs and indels in difficult-to-map regions from long-read sequencing by haplotype-aware deep neural networks

2019 ◽  
Author(s):  
Umair Ahsan ◽  
Qian Liu ◽  
Li Fang ◽  
Kai Wang
Keyword(s):  
Deep Neural Network ◽  
Deep Neural Networks ◽  
Variant Calling ◽  
Sequencing Data ◽  
Long Reads ◽  
Novel Variants ◽  
Long Read ◽  
Variant Detection ◽  
Genomic Regions ◽  
Haplotype Information

AbstractVariant (SNPs/indels) detection from high-throughput sequencing data remains an important yet unresolved problem. Long-read sequencing enables variant detection in difficult-to-map genomic regions that short-read sequencing cannot reliably examine (for example, only ~80% of genomic regions are marked as “high-confidence region” to have SNP/indel calls in the Genome In A Bottle project); however, the high per-base error rate poses unique challenges in variant detection. Existing methods on long-read data typically rely on analyzing pileup information from neighboring bases surrounding a candidate variant, similar to short-read variant callers, yet the benefits of much longer read length are not fully exploited. Here we present a deep neural network called NanoCaller, which detects SNPs by examining pileup information solely from other nonadjacent candidate SNPs that share the same long reads using long-range haplotype information. With called SNPs by NanoCaller, NanoCaller phases long reads and performs local realignment on two sets of phased reads to call indels by another deep neural network. Extensive evaluation on 5 human genomes (sequenced by Nanopore and PacBio long-read techniques) demonstrated that NanoCaller greatly improved performance in difficult-to-map regions, compared to other long-read variant callers. We experimentally validated 41 novel variants in difficult-to-map regions in a widely-used benchmarking genome, which cannot be reliably detected previously. We extensively evaluated the run-time characteristics and the sensitivity of parameter settings of NanoCaller to different characteristics of sequencing data. Finally, we achieved the best performance in Nanopore-based variant calling from MHC regions in the PrecisionFDA Variant Calling Challenge on Difficult-to-Map Regions by ensemble calling. In summary, by incorporating haplotype information in deep neural networks, NanoCaller facilitates the discovery of novel variants in complex genomic regions from long-read sequencing data.

scholarly journals Impact of Chimera-less Long Reads on Metagenomics of Human Gut Viromes Treated With Multiple Displacement Amplification

2020 ◽  
Author(s):  
Yuya Kiguchi ◽  
Suguru Nishijima ◽  
Naveen Kumar ◽  
Masahira Hattori ◽  
Wataru Suda
Keyword(s):  
De Novo ◽  
Multiple Displacement Amplification ◽  
Read Length ◽  
Human Gut ◽  
Average Read Length ◽  
Sequencing Technologies ◽  
Genomic Complexity ◽  
Long Reads ◽  
Long Read ◽  
The Impact

Abstract Background: The ecological and biological features of the indigenous phage community (virome) in the human gut microbiome are poorly understood, possibly due to many fragmented contigs and fewer complete genomes based on conventional short-read metagenomics. Long-read sequencing technologies have attracted attention as an alternative approach to reconstruct long and accurate contigs from microbial communities. However, the impact of long-read metagenomics on human gut virome analysis has not been well evaluated. Results: Here we present chimera-less PacBio long-read metagenomics of multiple displacement amplification (MDA)-treated human gut virome DNA. The method included the development of a novel bioinformatics tool, SACRA (Split Amplified Chimeric Read Algorithm), which efficiently detects and splits numerous chimeric reads in PacBio reads from the MDA-treated virome samples. SACRA treatment of PacBio reads from five samples markedly reduced the average chimera ratio from 72 to 1.5%, generating chimera-less PacBio reads with an average read-length of 1.8 kb. De novo assembly of the chimera-less long reads generated contigs with an average N50 length of 11.1 kb, whereas those of MiSeq short reads from the same samples were 0.7 kb, dramatically improving contig extension. Alignment of both contig sets generated 378 high-quality merged contigs (MCs) composed of the minimum scaffolds of 434 MiSeq and 637 PacBio contigs, respectively, and also identified numerous MiSeq short fragmented contigs ≤500 bp additionally aligned to MCs, which possibly originated from a small fraction of MiSeq chimeric reads. The alignment also revealed that fragmentations of the scaffolded MiSeq contigs were caused primarily by genomic complexity of the community, including local repeats, hypervariable regions, and highly conserved sequences in and between the phage genomes. We identified 142 complete and near-complete phage genomes including 108 novel genomes, varying from 5 to 185 kb in length, the majority of which were predicted to be Microviridae phages including several variants with homologous but distinct genomes, which were fragmented in MiSeq contigs. Conclusions: Long-read metagenomics coupled with SACRA provides an improved method to reconstruct accurate and extended phage genomes from MDA-treated virome samples of the human gut, and potentially from other environmental virome samples.

scholarly journals LongPhase: an ultra-fast chromosome-scale phasing algorithm for small and large variants

2021 ◽  
Author(s):  
Jyun-Hong Lin ◽  
Liang-Chi Chen ◽  
Shu-Qi Yu ◽  
Yao-Ting Huang
Keyword(s):  
Variant Calling ◽  
Cost Effective ◽  
Nucleotide Polymorphisms ◽  
Structural Variations ◽  
Single Nucleotide ◽  
Chromosome Conformation ◽  
Long Reads ◽  
Cost Effective Approach ◽  
Long Read ◽  
Microbial Strains

AbstractLong-read phasing has been used for reconstructing diploid genomes, improving variant calling, and resolving microbial strains in metagenomics. However, the phasing blocks of existing methods are broken by large Structural Variations (SVs), and the efficiency is unsatisfactory for population-scale phasing. This paper presents an ultra-fast algorithm, LongPhase, which can simultaneously phase single nucleotide polymorphisms (SNPs) and SVs of a human genome in ∼10-20 minutes, 10x faster than the state-of-the-art WhatsHap and Margin. In particular, LongPhase produces much larger phased blocks at almost chromosome level with only long reads (N50=26Mbp). We demonstrate that LongPhase combined with Nanopore is a cost-effective approach for providing chromosome-scale phasing without the need for additional trios, chromosome-conformation, and single-cell strand-seq data.

scholarly journals Long-read assemblies reveal structural diversity in genomes of organelles - an example with Acacia pycnantha

2020 ◽  
Author(s):  
Anna E. Syme ◽  
Todd G.B. McLay ◽  
Frank Udovicic ◽  
David J. Cantrill ◽  
Daniel J. Murphy
Keyword(s):  
Mitochondrial Genome ◽  
Chloroplast Genome ◽  
De Novo ◽  
Genomic Structure ◽  
Structural Diversity ◽  
Mitochondrial Genomes ◽  
Long Reads ◽  
Organelle Genomes ◽  
Long Read ◽  
Assembly Algorithms

AbstractAlthough organelle genomes are typically represented as single, static, circular molecules, there is evidence that the chloroplast genome exists in two structural haplotypes and that the mitochondrial genome can display multiple circular, linear or branching forms. We sequenced and assembled chloroplast and mitochondrial genomes of the Golden Wattle, Acacia pycnantha, using long reads, iterative baiting to extract organelle-only reads, and several assembly algorithms to explore genomic structure. Using a de novo assembly approach agnostic to previous hypotheses about structure, we found different assemblies revealed contrasting arrangements of genomic segments; a hypothesis supported by mapped reads spanning alternate paths.

scholarly journals Long-read amplicon denoising

2018 ◽  
Author(s):  
Venkatesh Kumar ◽  
Thomas Vollbrecht ◽  
Mark Chernyshev ◽  
Sanjay Mohan ◽  
Brian Hanst ◽  
...  
Keyword(s):  
Ground Truth ◽  
Amplicon Sequencing ◽  
Error Rates ◽  
Sequencing Error ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Medium Length ◽  
Long Reads ◽  
Long Read ◽  
Error Profiles

Long-read next generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies. Called “amplicon denoising”, this problem has been extensively studied for short-read sequencing technologies, but current solutions do not appear to generalize well to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads (here ~2.6kb) and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower.On one real dataset with ground truth, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method.Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD) are implemented purely in the Julia scientific computing language, and are hereby released along with a complete toolkit of functions that allow long-read amplicon sequence analysis pipelines to be constructed in pure Julia. Further, we make available a webserver to dramatically simplify the processing of long-read PacBio sequences.

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.

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