Contingency and selection in mitochondrial genome dynamics

Genome Dynamics ◽

Eukaryotic cells contain numerous copies of mitochondrial DNA (mtDNA), allowing for the coexistence of mutant and wild-type mtDNA in individual cells. The fate of mutant mtDNA depends on their relative replicative fitness within cells and the resulting cellular fitness within populations of cells. Yet the dynamics of the generation of mutant mtDNA and features that inform their fitness remain unaddressed. Here we utilize long read single-molecule sequencing to track mtDNA mutational trajectories in Saccharomyces cerevisiae. We show a previously unseen pattern that constrains subsequent excision events in mtDNA fragmentation. We also provide evidence for the generation of rare and contentious non-periodic mtDNA structures that lead to persistent diversity within individual cells. Finally, we show that measurements of relative fitness of mtDNA fit a phenomenological model that highlights important biophysical parameters governing mtDNA fitness. Altogether, our study provides techniques and insights into the dynamics of large structural changes in genomes that may be applicable in more complex organisms.

Analysis of the Complete Genome Sequence of a Novel, Pseudorabies Virus Strain Isolated in Southeast Europe

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2019/1806842 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12

Author(s):

Zsolt Csabai ◽

Dóra Tombácz ◽

Zoltán Deim ◽

Michael Snyder ◽

Zsolt Boldogkői

Keyword(s):

Single Molecule ◽

Base Composition ◽

Pseudorabies Virus ◽

Point Mutations ◽

Wild Type Virus ◽

Economic Losses ◽

Wild Type ◽

Southeast Europe ◽

Long Read ◽

National Programs

Background. Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease giving rise to significant economic losses worldwide. Many countries have implemented national programs for the eradication of this virus. In this study, long-read sequencing was used to determine the nucleotide sequence of the genome of a novel PRV strain (PRV-MdBio) isolated in Serbia.Results. In this study, a novel PRV strain was isolated and characterized. PRV-MdBio was found to exhibit similar growth properties to those of another wild-type PRV, the strain Kaplan. Single-molecule real-time (SMRT) sequencing has revealed that the new strain differs significantly in base composition even from strain Kaplan, to which it otherwise exhibits the highest similarity. We compared the genetic composition of PRV-MdBio to strain Kaplan and the China reference strain Ea and obtained that radical base replacements were the most common point mutations preceding conservative and silent mutations. We also found that the adaptation of PRV to cell culture does not lead to any tendentious genetic alteration in the viral genome.Conclusion. PRV-MdBio is a wild-type virus, which differs in base composition from other PRV strains to a relatively large extent.

Contig annotation tool CAT robustly classifies assembled metagenomic contigs and long sequences

10.1101/072868 ◽

2016 ◽

Cited By ~ 13

Author(s):

Diego D. Cambuy ◽

Felipe H. Coutinho ◽

Bas E. Dutilh

Keyword(s):

Single Molecule ◽

Dna Sequences ◽

Taxonomic Classification ◽

Annotation Tool ◽

Short Read ◽

Long Read ◽

Micro Organisms ◽

Taxonomic Annotation

AbstractIn modern-day metagenomics, there is an increasing need for robust taxonomic annotation of long DNA sequences from unknown micro-organisms. Long metagenomic sequences may be derived from assembly of short-read metagenomes, or from long-read single molecule sequencing. Here we introduce CAT, a pipeline for robust taxonomic classification of long DNA sequences. We show that CAT correctly classifies contigs at different taxonomic levels, even in simulated metagenomic datasets that are very distantly related from the sequences in the database. CAT is implemented in Python and the required scripts can be freely downloaded from Github.

Haplotype Diversity and Sequence Heterogeneity of Human Telomeres

10.1101/2020.01.31.929307 ◽

2020 ◽

Author(s):

Kirill Grigorev ◽

Jonathan Foox ◽

Daniela Bezdan ◽

Daniel Butler ◽

Jared J. Luxton ◽

...

Keyword(s):

Single Molecule ◽

Disease Risk ◽

Haplotype Diversity ◽

Sequence Heterogeneity ◽

Repetitive Nature ◽

Telomere Sequence ◽

Long Read ◽

Motif Composition ◽

Intractable Problem

AbstractTelomeres are regions of repetitive nucleotide sequences capping the ends of eukaryotic chromosomes that protect against deterioration, whose lengths can be correlated with age and disease risk factors. Given their length and repetitive nature, telomeric regions are not easily reconstructed from short read sequencing, making telomere sequence resolution a very costly and generally intractable problem. Recently, long-read sequencing, with read lengths measuring in hundreds of Kbp, has made it possible to routinely read into telomeric regions and inspect their structure. Here, we describe a framework for extracting telomeric reads from single-molecule sequencing experiments, describing their sequence variation and motifs, and for haplotype inference. We find that long telomeric stretches can be accurately captured with long-read sequencing, observe extensive sequence heterogeneity of human telomeres, discover and localize non-canonical motifs (both previously reported as well as novel), and report the first motif composition maps of human telomeric diplotypes on a multi-Kbp scale.

A whole genome atlas of 81 Psilocybe genomes as a resource for psilocybin production.

F1000Research ◽

10.12688/f1000research.55301.2 ◽

2021 ◽

Vol 10 ◽

pp. 961

Author(s):

Kevin McKernan ◽

Liam Kane ◽

Yvonne Helbert ◽

Lei Zhang ◽

Nathan Houde ◽

...

Keyword(s):

Gene Cluster ◽

Single Molecule ◽

Reference Genome ◽

De Novo ◽

Genomic Diversity ◽

Sequence Coverage ◽

Contiguous Gene ◽

Long Read ◽

Interesting Variation

The Psilocybe genus is well known for the synthesis of valuable psychoactive compounds such as Psilocybin, Psilocin, Baeocystin and Aeruginascin. The ubiquity of Psilocybin synthesis in Psilocybe has been attributed to a horizontal gene transfer mechanism of a ~20Kb gene cluster. A recently published highly contiguous reference genome derived from long read single molecule sequencing has underscored interesting variation in this Psilocybin synthesis gene cluster. This reference genome has also enabled the shotgun sequencing of spores from many Psilocybe strains to better catalog the genomic diversity in the Psilocybin synthesis pathway. Here we present the de novo assembly of 81 Psilocybe genomes compared to the P.envy reference genome. Surprisingly, the genomes of Psilocybe galindoi, Psilocybe tampanensis and Psilocybe azurescens lack sequence coverage over the previously described Psilocybin synthesis pathway but do demonstrate amino acid sequence homology to a less contiguous gene cluster and may illuminate the previously proposed evolution of psilocybin synthesis.

AsmMix: A pipeline for high quality diploid de novo assembly

10.1101/2021.01.15.426893 ◽

2021 ◽

Author(s):

Pei Wu ◽

Chao Liu ◽

Ou Wang ◽

Xia Zhao ◽

Fang Chen ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Variant Calling ◽

The Other ◽

Second Step ◽

Small Scale ◽

Mixing Process ◽

High Quality ◽

AbstractIn this paper, we report a pipeline, AsmMix, which is capable of producing both contiguous and high-quality diploid genomes. The pipeline consists of two steps. In the first step, two sets of assemblies are generated: one is based on co-barcoded reads, which are highly accurate and haplotype-resolved but contain many gaps, the other assembly is based on single-molecule sequencing reads, which is contiguous but error-prone. In the second step, those two sets of assemblies are compared and integrated into a haplotype-resolved assembly with fewer errors. We test our pipeline using a dataset of human genome NA24385, perform variant calling from those assemblies and then compare against GIAB Benchmark. We show that AsmMix pipeline could produce highly contiguous, accurate, and haplotype-resolved assemblies. Especially the assembly mixing process could effectively reduce small-scale errors in the long read assembly.

Mapping the epigenetic modifications of DNA and RNA

Protein & Cell ◽

10.1007/s13238-020-00733-7 ◽

2020 ◽

Vol 11 (11) ◽

pp. 792-808 ◽

Cited By ~ 6

Author(s):

Lin-Yong Zhao ◽

Jinghui Song ◽

Yibin Liu ◽

Chun-Xiao Song ◽

Chengqi Yi

Keyword(s):

Single Molecule ◽

Detection Methods ◽

Rna Modification ◽

Rna Modifications ◽

Dna And Rna ◽

Third Generation Sequencing ◽

Technological Advances ◽

Long Read ◽

High Throughput Detection

Abstract Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

Detecting AGG Interruptions in Females With a FMR1 Premutation by Long-Read Single-Molecule Sequencing: A 1 Year Clinical Experience

Frontiers in Genetics ◽

10.3389/fgene.2018.00150 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 6

Author(s):

Simon Ardui ◽

Valerie Race ◽

Thomy de Ravel ◽

Hilde Van Esch ◽

Koenraad Devriendt ◽

...

Keyword(s):

Clinical Experience ◽

Single Molecule ◽

Fmr1 Premutation ◽

Long Read ◽

Agg Interruptions

Resolving the Complexity of Human Skin Metagenomes Using Single-Molecule Sequencing

mBio ◽

10.1128/mbio.01948-15 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 37

Author(s):

Yu-Chih Tsai ◽

Sean Conlan ◽

Clayton Deming ◽

Julia A. Segre ◽

Heidi H. Kong ◽

...

Keyword(s):

Microbial Community ◽

Human Skin ◽

Single Molecule ◽

Smrt Sequencing ◽

High Quality ◽

Single Nucleotide ◽

Short Read ◽

Hybrid Approaches ◽

ABSTRACT Deep metagenomic shotgun sequencing has emerged as a powerful tool to interrogate composition and function of complex microbial communities. Computational approaches to assemble genome fragments have been demonstrated to be an effective tool for de novo reconstruction of genomes from these communities. However, the resultant “genomes” are typically fragmented and incomplete due to the limited ability of short-read sequence data to assemble complex or low-coverage regions. Here, we use single-molecule, real-time (SMRT) sequencing to reconstruct a high-quality, closed genome of a previously uncharacterized Corynebacterium simulans and its companion bacteriophage from a skin metagenomic sample. Considerable improvement in assembly quality occurs in hybrid approaches incorporating short-read data, with even relatively small amounts of long-read data being sufficient to improve metagenome reconstruction. Using short-read data to evaluate strain variation of this C. simulans in its skin community at single-nucleotide resolution, we observed a dominant C. simulans strain with moderate allelic heterozygosity throughout the population. We demonstrate the utility of SMRT sequencing and hybrid approaches in metagenome quantitation, reconstruction, and annotation. IMPORTANCE The species comprising a microbial community are often difficult to deconvolute due to technical limitations inherent to most short-read sequencing technologies. Here, we leverage new advances in sequencing technology, single-molecule sequencing, to significantly improve reconstruction of a complex human skin microbial community. With this long-read technology, we were able to reconstruct and annotate a closed, high-quality genome of a previously uncharacterized skin species. We demonstrate that hybrid approaches with short-read technology are sufficiently powerful to reconstruct even single-nucleotide polymorphism level variation of species in this a community.

Genetic Findings of Sanger and Nanopore Single-Molecule Sequencing in Patients with X-Linked Hearing Loss and Incomplete Partition Type III

10.21203/rs.3.rs-501574/v1 ◽

2021 ◽

Author(s):

Ying Chen ◽

Jiajun Qiu ◽

Yingwei Wu ◽

Huan Jia ◽

Yi Jiang ◽

...

Keyword(s):

Single Molecule ◽

Hearing Aids ◽

Sanger Sequencing ◽

Clinical Characteristics ◽

De Novo ◽

Genetic Mutations ◽

Long Read ◽

Incomplete Partition ◽

The Mean

Abstract BackgroundPOU3F4 is the causative gene for X-linked deafness-2 (DFNX2), characterized by incomplete partition type III (IP-III) malformation of the inner ear. The aim of this study was to investigate the clinical characteristics and molecular findings by Sanger or Nanopore single-molecule sequencing in IP-III patients. MethodsDiagnosis of IP-III was mainly based on clinical characteristics including radiological and audiological findings. Sanger sequencing of POU3F4 were carried out for these IP-III patients. For those patients with negative results for POU3F4 Sanger sequencing, Nanopore long-read single-molecule sequencing was used to identify the possible pathogenic variants. Hearing intervention outcomes of hearing aids fitting and cochlear implantation were also analyzed. Grouped by different locations of POU3F4 variants, aided PTA was further compared between patients in whom the variants located in the exon region or in the upstream region.ResultsIn total, 18 male patients from 14 unrelated families were diagnosed with IP-III. 10 variants were identified in POU3F4 by Sanger sequencing and 9 of these were novel (p.Val321Gly, p.Gln181*, p.Cys233*, p.Val215Gly, p.Arg282Gln, p.Trp57*, p.Gln316*, c.903_912 delins TGCCA and p.Arg205del). Four different deletions (DELs) that varied from 80 to 486 kb were identified 876-1503 kb upstream of POU3F4 by Nanopore long-read single-molecule sequencing. Of them, de novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutations. Of these 18 patients, 7 had bilateral hearing aids (HAs) and 10 patients received unilateral cochlear implantation (CI). The mean aided pure tone average (PTA) for HAs and CI users were 41.1±5.18 and 40.3±7.59 dB HL respectively. The mean PTAs for whom the variants located in the exon and upstream regions were 39.6±6.31 vs 43.0±7.10 dB HL, which presented no significant difference (p=0.342).ConclusionsAmong IP-III patients, 28.6% (4/14) had no definite mutation in exon region of POU3F4, however, possible pathogenic deletions were identified in upstream region of this gen. De novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutation. Hearing intervention outcomes of IP-III patients presented no difference regardless of the variants locations on exon or upstream regions.

Assembling Large Genomes with Single-Molecule Sequencing and Locality Sensitive Hashing

10.1101/008003 ◽

2014 ◽

Cited By ~ 13

Author(s):

Konstantin Berlin ◽

Sergey Koren ◽

Chen-Shan Chin ◽

James Drake ◽

Jane M Landolin ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Locality Sensitive Hashing ◽

Model Organisms ◽

Smrt Sequencing ◽

High Coverage ◽

Celera Assembler ◽

Long Reads ◽

We report reference-grade de novo assemblies of four model organisms and the human genome from single-molecule, real-time (SMRT) sequencing. Long-read SMRT sequencing is routinely used to finish microbial genomes, but the available assembly methods have not scaled well to larger genomes. Here we introduce the MinHash Alignment Process (MHAP) for efficient overlapping of noisy, long reads using probabilistic, locality-sensitive hashing. Together with Celera Assembler, MHAP was used to reconstruct the genomes of Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, and human from high-coverage SMRT sequencing. The resulting assemblies include fully resolved chromosome arms and close persistent gaps in these important reference genomes, including heterochromatic and telomeric transition sequences. For D. melanogaster, MHAP achieved a 600-fold speedup relative to prior methods and a cloud computing cost of a few hundred dollars. These results demonstrate that single-molecule sequencing alone can produce near-complete eukaryotic genomes at modest cost.