scholarly journals What is in a lichen? A metagenomic approach to reconstruct the holo-genome of Umbilicaria pustulata

2019 ◽  
Author(s):  
Bastian Greshake Tzovaras ◽  
Francisca H.I.D. Segers ◽  
Anne Bicker ◽  
Francesco Dal Grande ◽  
Jürgen Otte ◽  
...  
Keyword(s):  
Single Species ◽  
Fungal Genome ◽  
Sequencing Error ◽  
Associated Bacteria ◽  
Long Reads ◽  
Missing Gene ◽  
Symbiosis Research ◽  
Genome Assemblies ◽  
Algal Chloroplast ◽  
Metagenomic Approach

AbstractLichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are generally poor candidates for experimentation. Obtaining contiguous, high quality genomes for such symbiotic communities is technically challenging. Here we present the first assembly of a lichen holo-genome from metagenomic whole genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis – the fungus Umbilicaria pustulata (33 Mbp) and the green alga Trebouxia sp. (53 Mbp) – were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative cellular abundance of approximately 20:1 (fungus:alga). Gap-free, circular sequences for all organellar genomes were obtained. The community of lichen-associated bacteria is dominated by Acidobacteriaceae, and the two largest bacterial contigs belong to the genus Acidobacterium. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that G/C-rich inverted repeats paired with non-random sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.

scholarly journals What Is in Umbilicaria pustulata? A Metagenomic Approach to Reconstruct the Holo-Genome of a Lichen

2020 ◽  
Vol 12 (4) ◽  
pp. 309-324 ◽  
Author(s):  
Bastian Greshake Tzovaras ◽  
Francisca H I D Segers ◽  
Anne Bicker ◽  
Francesco Dal Grande ◽  
Jürgen Otte ◽  
...  
Keyword(s):  
Single Species ◽  
Fungal Genome ◽  
Sequencing Error ◽  
Organellar Genomes ◽  
Long Reads ◽  
Missing Gene ◽  
Symbiosis Research ◽  
Genome Assemblies ◽  
Algal Chloroplast ◽  
Metagenomic Approach

Abstract Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis—the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)—were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of ∼20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.

scholarly journals Chromosome-level hybrid de novo genome assemblies as an attainable option for non-model organisms

2019 ◽  
Author(s):  
Coline C. Jaworski ◽  
Carson W. Allan ◽  
Luciano M. Matzkin
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Model Organism ◽  
Model Organisms ◽  
Sequencing Error ◽  
Long Reads ◽  
Hybrid Genome ◽  
Genome Assemblies ◽  
Hybrid Assemblies ◽  
Chromosome Level

AbstractThe emergence of third generation sequencing (3GS; long-reads) is making closer the goal of chromosome-size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of non-model organisms. However, long-read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short-reads and long-reads provide an alternative efficient and cost-effective approach to generate de novo, chromosome-level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation is constantly being expanded and improved. This makes it difficult for non-experts to find efficient, fast and tractable computational solutions for genome assembly, especially in the case of non-model organisms lacking a reference genome or one from a closely related species. In this study, we review and test the most recent pipelines for hybrid assemblies, comparing the model organism Drosophila melanogaster to a non-model cactophilic Drosophila, D. mojavensis. We show that it is possible to achieve excellent contiguity on this non-model organism using the DBG2OLC pipeline.

scholarly journals Hapo-G, haplotype-aware polishing of genome assemblies with accurate reads

2021 ◽  
Vol 3 (2) ◽  
Author(s):  
Jean-Marc Aury ◽  
Benjamin Istace
Keyword(s):  
Single Molecule ◽  
Direct Consequence ◽  
High Quality ◽  
Sequencing Errors ◽  
Coding Regions ◽  
Sequencing Technologies ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
Genome Assemblies

Abstract 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 high-quality reads (short or long-reads) to polish genome assemblies and in particular assemblies of diploid and heterozygous genomes.

scholarly journals A highly contiguous nuclear genome assembly of the mandarinfish Synchiropus splendidus (Syngnathiformes: Callionymidae)

2021 ◽  
Author(s):  
Martin Stervander ◽  
William A Cresko
Keyword(s):  
Genome Assembly ◽  
Nuclear Genome ◽  
Phylogenetic Position ◽  
Blue Color ◽  
Long Reads ◽  
The Family ◽  
Commercially Important ◽  
Genomic Resource ◽  
Genome Assemblies ◽  
Important Fish

Abstract The fish order Syngnathiformes has been referred to as a collection of misfit fishes, comprising commercially important fish such as red mullets as well as the highly diverse seahorses, pipefishes, and seadragons—the well-known family Syngnathidae, with their unique adaptations including male pregnancy. Another ornate member of this order is the species mandarinfish. No less than two types of chromatophores have been discovered in the spectacularly colored mandarinfish: the cyanophore (producing blue color) and the dichromatic cyano-erythrophore (producing blue and red). The phylogenetic position of mandarinfish in Syngnathiformes, and their promise of additional genetic discoveries beyond the chromatophores, made mandarinfish an appealing target for whole genome sequencing. We used linked sequences to create synthetic long reads, producing a highly contiguous genome assembly for the mandarinfish. The genome assembly comprises 483 Mbp (longest scaffold 29 Mbp), has an N50 of 12 Mbp, and an L50 of 14 scaffolds. The assembly completeness is also high, with 92.6% complete, 4.4% fragmented, and 2.9% missing out of 4,584 BUSCO genes found in ray-finned fishes. Outside the family Syngnathidae, the mandarinfish represents one of the most contiguous syngnathiform genome assemblies to date. The mandarinfish genomic resource will likely serve as a high-quality outgroup to syngnathid fish, and furthermore for research on the genomic underpinnings of the evolution of novel pigmentation.

scholarly journals Single-molecule sequencing and conformational capture enable de novo mammalian reference genomes

2016 ◽  
Author(s):  
Derek M. Bickhart ◽  
Benjamin D. Rosen ◽  
Sergey Koren ◽  
Brian L. Sayre ◽  
Alex R. Hastie ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Chromosome Length ◽  
Chromatin Interaction ◽  
Capra Hircus ◽  
Immune Gene ◽  
Ruminant Species ◽  
Long Reads ◽  
Assembly Algorithms ◽  
Genome Assemblies

AbstractThe decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus), based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced the most contiguous de novo mammalian assembly to date, with chromosome-length scaffolds and only 663 gaps. Our assembly represents a >250-fold improvement in contiguity compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, supporting the most complete repeat family and immune gene complex representation ever produced for a ruminant species.

scholarly journals Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Caroline Belser ◽  
Franc-Christophe Baurens ◽  
Benjamin Noel ◽  
Guillaume Martin ◽  
Corinne Cruaud ◽  
...  
Keyword(s):  
Musa Acuminata ◽  
Genetic Maps ◽  
Nanopore Sequencing ◽  
Genome Coverage ◽  
Long Reads ◽  
Oxford Nanopore ◽  
A Genome ◽  
Long Read ◽  
Genome Assemblies ◽  
First Time

AbstractLong-read technologies hold the promise to obtain more complete genome assemblies and to make them easier. Coupled with long-range technologies, they can reveal the architecture of complex regions, like centromeres or rDNA clusters. These technologies also make it possible to know the complete organization of chromosomes, which remained complicated before even when using genetic maps. However, generating a gapless and telomere-to-telomere assembly is still not trivial, and requires a combination of several technologies and the choice of suitable software. Here, we report a chromosome-scale assembly of a banana genome (Musa acuminata) generated using Oxford Nanopore long-reads. We generated a genome coverage of 177X from a single PromethION flowcell with near 17X with reads longer than 75 kbp. From the 11 chromosomes, 5 were entirely reconstructed in a single contig from telomere to telomere, revealing for the first time the content of complex regions like centromeres or clusters of paralogous genes.

scholarly journals Factorial estimating assembly base errors using k-mer abundance difference (KAD) between short reads and genome assembled sequences

2020 ◽  
Vol 2 (3) ◽  
Author(s):  
Cheng He ◽  
Guifang Lin ◽  
Hairong Wei ◽  
Haibao Tang ◽  
Frank F White ◽  
...  
Keyword(s):  
Copy Number ◽  
Error Rates ◽  
Genome Sequences ◽  
Short Reads ◽  
Sequencing Technologies ◽  
Insertion And Deletion ◽  
Novel Approach ◽  
Long Reads ◽  
Long Read ◽  
Genome Assemblies

Abstract Genome sequences provide genomic maps with a single-base resolution for exploring genetic contents. Sequencing technologies, particularly long reads, have revolutionized genome assemblies for producing highly continuous genome sequences. However, current long-read sequencing technologies generate inaccurate reads that contain many errors. Some errors are retained in assembled sequences, which are typically not completely corrected by using either long reads or more accurate short reads. The issue commonly exists, but few tools are dedicated for computing error rates or determining error locations. In this study, we developed a novel approach, referred to as k-mer abundance difference (KAD), to compare the inferred copy number of each k-mer indicated by short reads and the observed copy number in the assembly. Simple KAD metrics enable to classify k-mers into categories that reflect the quality of the assembly. Specifically, the KAD method can be used to identify base errors and estimate the overall error rate. In addition, sequence insertion and deletion as well as sequence redundancy can also be detected. Collectively, KAD is valuable for quality evaluation of genome assemblies and, potentially, provides a diagnostic tool to aid in precise error correction. KAD software has been developed to facilitate public uses.

scholarly journals LINKS: Scaffolding genome assemblies with kilobase-long nanopore reads

2015 ◽  
Author(s):  
Rene L Warren ◽  
Benjamin P Vandervalk ◽  
Steven JM Jones ◽  
Inanc Birol
Keyword(s):  
Genome Assembly ◽  
Error Rates ◽  
Great Promise ◽  
Genomic Information ◽  
E Coli ◽  
Long Reads ◽  
Oxford Nanopore ◽  
Long Read ◽  
K 12 ◽  
Genome Assemblies

Owing to the complexity of the assembly problem, we do not yet have complete genome sequences. The difficulty in assembling reads into finished genomes is exacerbated by sequence repeats and the inability of short reads to capture sufficient genomic information to resolve those problematic regions. Established and emerging long read technologies show great promise in this regard, but their current associated higher error rates typically require computational base correction and/or additional bioinformatics pre-processing before they could be of value. We present LINKS, the Long Interval Nucleotide K-mer Scaffolder algorithm, a solution that makes use of the information in error-rich long reads, without the need for read alignment or base correction. We show how the contiguity of an ABySS E. coli K-12 genome assembly could be increased over five-fold by the use of beta-released Oxford Nanopore Ltd. (ONT) long reads and how LINKS leverages long-range information in S. cerevisiae W303 ONT reads to yield an assembly with less than half the errors of competing applications. Re-scaffolding the colossal white spruce assembly draft (PG29, 20 Gbp) and how LINKS scales to larger genomes is also presented. We expect LINKS to have broad utility in harnessing the potential of long reads in connecting high-quality sequences of small and large genome assembly drafts. Availability: http://www.bcgsc.ca/bioinfo/software/links

SGTK: a toolkit for visualization and assessment of scaffold graphs

2018 ◽  
Vol 35 (13) ◽  
pp. 2303-2305 ◽  
Author(s):  
Olga Kunyavskaya ◽  
Andrey D Prjibelski
Keyword(s):  
Software Package ◽  
Supplementary Information ◽  
Sequencing Data ◽  
Software Developers ◽  
Long Reads ◽  
Mate Pair ◽  
Linkage Information ◽  
Assembly Pipeline ◽  
Genome Assemblies ◽  
Assembly Software

Abstract Summary Scaffolding is an important step in every genome assembly pipeline, which allows to order contigs into longer sequences using various types of linkage information, such as mate-pair libraries and long reads. In this work, we operate with a notion of a scaffold graph—a graph, vertices of which correspond to the assembled contigs and edges represent connections between them. We present a software package called Scaffold Graph ToolKit that allows to construct and visualize scaffold graphs using different kinds of sequencing data. We show that the scaffold graph appears to be useful for analyzing and assessing genome assemblies, and demonstrate several use cases that can be helpful for both assembly software developers and their users. Availability and implementation SGTK is implemented in C++, Python and JavaScript and is freely available at https://github.com/olga24912/SGTK. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Assembly of chromosome-scale contigs by efficiently resolving repetitive sequences with long reads

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Huilong Du ◽  
Chengzhi Liang
Keyword(s):  
Single Molecule ◽  
Repetitive Sequences ◽  
Tartary Buckwheat ◽  
Gene Sequences ◽  
Assembly Method ◽  
Long Reads ◽  
A Genome ◽  
Genome Assemblies ◽  
Reference Genomes

AbstractThe abundant repetitive sequences in complex eukaryotic genomes cause fragmented assemblies, which lose value as reference genomes, often due to incomplete gene sequences and unanchored or mispositioned contigs on chromosomes. Here we report a genome assembly method HERA, which resolves repeats efficiently by constructing a connection graph from an overlap graph. We test HERA on the genomes of rice, maize, human, and Tartary buckwheat with single-molecule sequencing and mapping data. HERA correctly assembles most of the previously unassembled regions, resulting in dramatically improved, highly contiguous genome assemblies with newly assembled gene sequences. For example, the maize contig N50 size reaches 61.2 Mb and the Tartary buckwheat genome comprises only 20 contigs. HERA can also be used to fill gaps and fix errors in reference genomes. The application of HERA will greatly improve the quality of new or existing assemblies of complex genomes.

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