scholarly journals Genomic sequence characteristics and the empiric accuracy of short-read sequencing

2021 ◽  
Author(s):  
Maximillian G Marin ◽  
Roger Vargas ◽  
Michael Harris ◽  
Brendan Jeffrey ◽  
L. Elaine Epperson ◽  
...  
Keyword(s):  
Genomic Sequence ◽  
Repetitive Sequences ◽  
Gc Content ◽  
Variant Calling ◽  
Basic Research ◽  
Confidence Regions ◽  
Surveillance Systems ◽  
Short Read ◽  
Short Read Alignment ◽  
Long Reads

Background: Short-read whole genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences, and sequencing bias, reduce the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. For the clonal pathogen Mycobacterium tuberculosis (Mtb), researchers frequently exclude 10.7% of the genome believed to be repetitive and prone to erroneous variant calls. To benchmark short-read variant calling, we used 36 diverse clinical Mtb isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically study the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias, and GC content. Results: Reference based Illumina variant calling had a recall ≥89.0% and precision ≥98.5% across parameters evaluated. The best balance between precision and recall was achieved by tuning the mapping quality (MQ) threshold, i.e. confidence of the read mapping (recall 85.8%, precision 99.1% at MQ ≥ 40). Masking repetitive sequence content is an alternative conservative approach to variant calling that maintains high precision (recall 70.2%, precision 99.6% at MQ≥40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52 of the 168 PE/PPE genes (34.5%). We present a refined list of low confidence regions and examine the largest sources of variant calling error. Conclusions: Our improved approach to variant calling has broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems, and more generally for WGS applications in other organisms.

scholarly journals Expanding an expanded genome: long-read sequencing ofTrypanosoma cruzi

2018 ◽  
Author(s):  
Luisa Berná ◽  
Matías Rodríguez ◽  
María Laura Chiribao ◽  
Adriana Parodi-Talice ◽  
Sebastián Pita ◽  
...  
Keyword(s):  
Repetitive Sequences ◽  
Gc Content ◽  
Gene Copy ◽  
Accurate Estimation ◽  
Sequencing Technologies ◽  
Copy Numbers ◽  
Long Reads ◽  
Long Read ◽  
Large Clusters ◽  
Gene Copy Numbers

Although the genome ofTrypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degree of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated withT. cruzi´sgenome since they permit directly determining the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, allows not only accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of twoT. cruziclones: the hybrid TCC (DTU TcVI) and the non-hybrid Dm28c (DTU TcI), determined by PacBio SMRT technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome ofT. cruziis composed of a "core compartment" and a "disruptive compartment" which exhibit opposite gene and GC content composition. New tandem and disperse repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families MUC and trans-sialidases allows now a better overview of these complex groups of genes.

scholarly journals Economic Genome Assembly from Low Coverage Illumina and Nanopore Data

2020 ◽  
Author(s):  
Thomas Gatter ◽  
Sarah von Löhneysen ◽  
Polina Drozdova ◽  
Tom Hartmann ◽  
Peter F. Stadler
Keyword(s):  
Genomic Sequence ◽  
State Of The Art ◽  
Fruit Fly ◽  
Computational Effort ◽  
Maximum Weight ◽  
New Approach ◽  
Short Read ◽  
Long Reads ◽  
Long Read ◽  
Low Coverage

AbstractWe describe a new approach to assemble genomes from a combination of low-coverage short and long reads. LazyBastard starts from a bipartite overlap graph between long reads and restrictively filtered short-read unitigs, which are then reduced to a long-read overlap graph G. Edges are removed from G to obtain first a consistent orientation and then a DAG. Using heuristics based on properties of proper interval graphs, contigs are extracted as maximum weight paths. These are translated into genomic sequence only in the final step. A prototype implementation of LazyBastard, entirely written in python, not only yields significantly more accurate assemblies of the yeast and fruit fly genomes compared to state-of-the-art pipelines but also requires much less computational effort.FundingRSF / Helmholtz Association 18-44-06201; Deutsche Academische Austauschdienst, DFG STA 850/19-2 within SPP 1738; German Federal Ministery of Education an Research 031A538A, de.NBI-RBC

scholarly journals Ratatosk – Hybrid error correction of long reads enables accurate variant calling and assembly

2020 ◽  
Author(s):  
Guillaume Holley ◽  
Doruk Beyter ◽  
Helga Ingimundardottir ◽  
Snædis Kristmundsdottir ◽  
Hannes P. Eggertsson ◽  
...  
Keyword(s):  
Error Correction ◽  
Error Rate ◽  
Variant Calling ◽  
Read Length ◽  
De Bruijn Graph ◽  
Nucleotide Polymorphisms ◽  
Short Read ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Median Error

AbstractMotivationLong Read Sequencing (LRS) technologies are becoming essential to complement Short Read Sequencing (SRS) technologies for routine whole genome sequencing. LRS platforms produce DNA fragment reads, from 103 to 106 bases, allowing the resolution of numerous uncertainties left by SRS reads for genome reconstruction and analysis. In particular, LRS characterizes long and complex structural variants undetected by SRS due to short read length. Furthermore, assemblies produced with LRS reads are considerably more contiguous than with SRS while spanning previously inaccessible telomeric and centromeric regions. However, a major challenge to LRS reads adoption is their much higher error rate than SRS of up to 15%, introducing obstacles in downstream analysis pipelines.ResultsWe present Ratatosk, a new error correction method for erroneous long reads based on a compacted and colored de Bruijn graph built from accurate short reads. Short and long reads color paths in the graph while vertices are annotated with candidate Single Nucleotide Polymorphisms. Long reads are subsequently anchored to the graph using exact and inexact fc-mer matches to find paths corresponding to corrected sequences. We demonstrate that Ratatosk can reduce the raw error rate of Oxford Nanopore reads 6-fold on average with a median error rate as low as 0.28%. Ratatosk corrected data maintain nearly 99% accurate SNP calls and increase indel call accuracy by up to about 40% compared to the raw data. An assembly of the Ashkenazi individual HG002 created from Ratatosk corrected Oxford Nanopore reads yields a contig N50 of 43.22 Mbp and less misassemblies than an assembly created from PacBio HiFi reads.Availabilityhttps://github.com/DecodeGenetics/[email protected]

scholarly journals Extreme enrichment of VNTR-associated polymorphicity in human subtelomeres: genes with most VNTRs are predominantly expressed in the brain

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jasper Linthorst ◽  
Wim Meert ◽  
Matthew S. Hestand ◽  
Jonas Korlach ◽  
Joris Robert Vermeesch ◽  
...  
Keyword(s):  
Single Molecule ◽  
Neurological Disorders ◽  
Tandem Repeats ◽  
De Novo ◽  
Repetitive Sequences ◽  
Healthy Woman ◽  
Gc Content ◽  
Phenotypic Traits ◽  
Short Read ◽  
The Brain

Abstract The human genome harbors numerous structural variants (SVs) which, due to their repetitive nature, are currently underexplored in short-read whole-genome sequencing approaches. Using single-molecule, real-time (SMRT) long-read sequencing technology in combination with FALCON-Unzip, we generated a de novo assembly of the diploid genome of a 115-year-old Dutch cognitively healthy woman. We combined this assembly with two previously published haploid assemblies (CHM1 and CHM13) and the GRCh38 reference genome to create a compendium of SVs that occur across five independent human haplotypes using the graph-based multi-genome aligner REVEAL. Across these five haplotypes, we detected 31,680 euchromatic SVs (>50 bp). Of these, ~62% were comprised of repetitive sequences with ‘variable number tandem repeats’ (VNTRs), ~10% were mobile elements (Alu, L1, and SVA), while the remaining variants were inversions and indels. We observed that VNTRs with GC-content >60% and repeat patterns longer than 15 bp were 21-fold enriched in the subtelomeric regions (within 5 Mb of the ends of chromosome arms). VNTR lengths can expand to exceed a critical length which is associated with impaired gene transcription. The genes that contained most VNTRs, of which PTPRN2 and DLGAP2 are the most prominent examples, were found to be predominantly expressed in the brain and associated with a wide variety of neurological disorders. Repeat-induced variation represents a sizeable fraction of the genetic variation in human genomes and should be included in investigations of genetic factors associated with phenotypic traits, specifically those associated with neurological disorders. We make available the long and short-read sequence data of the supercentenarian genome, and a compendium of SVs as identified across 5 human haplotypes.

scholarly journals Ratatosk: hybrid error correction of long reads enables accurate variant calling and assembly

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Guillaume Holley ◽  
Doruk Beyter ◽  
Helga Ingimundardottir ◽  
Peter L. Møller ◽  
Snædis Kristmundsdottir ◽  
...  
Keyword(s):  
Error Correction ◽  
Human Genome ◽  
Error Rate ◽  
Variant Calling ◽  
High Error Rate ◽  
Sequencing Data ◽  
Short Read ◽  
Long Reads ◽  
Median Error ◽  
Long Read

AbstractA major challenge to long read sequencing data is their high error rate of up to 15%. We present Ratatosk, a method to correct long reads with short read data. We demonstrate on 5 human genome trios that Ratatosk reduces the error rate of long reads 6-fold on average with a median error rate as low as 0.22 %. SNP calls in Ratatosk corrected reads are nearly 99 % accurate and indel calls accuracy is increased by up to 37 %. An assembly of Ratatosk corrected reads from an Ashkenazi individual yields a contig N50 of 45 Mbp and less misassemblies than a PacBio HiFi reads assembly.

scholarly journals Evaluating the accuracy of Listeria monocytogenes assemblies from quasimetagenomic samples using long and short reads

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Seth Commichaux ◽  
Kiran Javkar ◽  
Padmini Ramachandran ◽  
Niranjan Nagarajan ◽  
Denis Bertrand ◽  
...  
Keyword(s):  
Public Health ◽  
Public Health Response ◽  
High Quality ◽  
Short Read ◽  
Short Reads ◽  
The Core ◽  
Long Reads ◽  
Health Response ◽  
Long Read ◽  
Core Genes

Abstract Background Whole genome sequencing of cultured pathogens is the state of the art public health response for the bioinformatic source tracking of illness outbreaks. Quasimetagenomics can substantially reduce the amount of culturing needed before a high quality genome can be recovered. Highly accurate short read data is analyzed for single nucleotide polymorphisms and multi-locus sequence types to differentiate strains but cannot span many genomic repeats, resulting in highly fragmented assemblies. Long reads can span repeats, resulting in much more contiguous assemblies, but have lower accuracy than short reads. Results We evaluated the accuracy of Listeria monocytogenes assemblies from enrichments (quasimetagenomes) of naturally-contaminated ice cream using long read (Oxford Nanopore) and short read (Illumina) sequencing data. Accuracy of ten assembly approaches, over a range of sequencing depths, was evaluated by comparing sequence similarity of genes in assemblies to a complete reference genome. Long read assemblies reconstructed a circularized genome as well as a 71 kbp plasmid after 24 h of enrichment; however, high error rates prevented high fidelity gene assembly, even at 150X depth of coverage. Short read assemblies accurately reconstructed the core genes after 28 h of enrichment but produced highly fragmented genomes. Hybrid approaches demonstrated promising results but had biases based upon the initial assembly strategy. Short read assemblies scaffolded with long reads accurately assembled the core genes after just 24 h of enrichment, but were highly fragmented. Long read assemblies polished with short reads reconstructed a circularized genome and plasmid and assembled all the genes after 24 h enrichment but with less fidelity for the core genes than the short read assemblies. Conclusion The integration of long and short read sequencing of quasimetagenomes expedited the reconstruction of a high quality pathogen genome compared to either platform alone. A new and more complete level of information about genome structure, gene order and mobile elements can be added to the public health response by incorporating long read analyses with the standard short read WGS outbreak response.

scholarly journals Chromosome-level de novo assembly of Coprinopsis cinerea A43mut B43mut pab1-1 #326 and genetic variant identification of mutants using Nanopore MinION sequencing

2020 ◽  
Author(s):  
Yichun Xie ◽  
Yiyi Zhong ◽  
Jinhui Chang ◽  
Hoi Shan Kwan
Keyword(s):  
Genome Assembly ◽  
Dna Isolation ◽  
Variant Calling ◽  
Genomic Analysis ◽  
Single Nucleotide ◽  
Sequencing Platform ◽  
Genomic Dna Isolation ◽  
Long Reads ◽  
Dna Isolation Protocol ◽  
Chromosome Level

AbstractThe homokaryotic Coprinopsis cinerea strain A43mut B43mut pab1-1 #326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factors A and B result in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with the C. cinerea strain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein (RasGAP) was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform.HighlightA chromosome-level genome assembly of C. cinerea #326A fast and efficient high-molecular-weight fungal genomic DNA isolation protocolStructural variant and single nucleotide variant calling using Nanopore readsA series of solutions and reference parameters for fungal genomic analysis on MinION

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 Fuzzy set intersection based paired-end short-read alignment

2021 ◽  
Author(s):  
William J Bolosky ◽  
Arun Subramaniyan ◽  
Matei Zaharia ◽  
Ravi Pandya ◽  
Taylor Sittler ◽  
...  
Keyword(s):  
Fuzzy Set ◽  
Genetic Material ◽  
Genomic Data ◽  
Short Read ◽  
Read Alignment ◽  
Short Read Alignment ◽  
Set Intersection

Much genomic data comes in the form of paired-end reads: two reads that represent genetic material with a small gap between. We present a new algorithm for aligning both reads in a pair simultaneously by fuzzily intersecting the sets of candidate alignment locations for each read. This algorithm is often much faster and produces alignments that result in variant calls having roughly the same concordance as the best competing aligners.

scholarly journals Rapid Mycobacterium tuberculosis spoligotyping from uncorrected long reads using Galru

2020 ◽  
Author(s):  
Andrew J. Page ◽  
Nabil-Fareed Alikhan ◽  
Michael Strinden ◽  
Thanh Le Viet ◽  
Timofey Skvortsov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
State Of The Art ◽  
Sequence Data ◽  
Human Pathogen ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Reads ◽  
Long Read

AbstractSpoligotyping of Mycobacterium tuberculosis provides a subspecies classification of this major human pathogen. Spoligotypes can be predicted from short read genome sequencing data; however, no methods exist for long read sequence data such as from Nanopore or PacBio. We present a novel software package Galru, which can rapidly detect the spoligotype of a Mycobacterium tuberculosis sample from as little as a single uncorrected long read. It allows for near real-time spoligotyping from long read data as it is being sequenced, giving rapid sample typing. We compare it to the existing state of the art software and find it performs identically to the results obtained from short read sequencing data. Galru is freely available from https://github.com/quadram-institute-bioscience/galru under the GPLv3 open source licence.

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