scholarly journals Perspectives and benefits of high-throughput long-read sequencing in microbial ecology

2021 ◽  
Author(s):  
Leho Tedersoo ◽  
Mads Albertsen ◽  
Sten Anslan ◽  
Benjamin Callahan
Keyword(s):  
Microbial Ecology ◽  
High Throughput ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Environmental Dna ◽  
Nanopore Sequencing ◽  
High Quality ◽  
Short Read ◽  
Sequencing Technologies ◽  
Long Read

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities such as rapid molecular diagnostics and direct RNA sequencing, and both PacBio and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

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 Mapping and phasing of structural variation in patient genomes using nanopore sequencing

2017 ◽  
Author(s):  
Mircea Cretu Stancu ◽  
Markus J. van Roosmalen ◽  
Ivo Renkens ◽  
Marleen Nieboer ◽  
Sjors Middelkamp ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Structural Variants ◽  
Human Genetic Disease ◽  
Structural Genomic ◽  
Short Read ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
Long Read ◽  
Complex Structural

AbstractStructural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications < 200bp in size. Long sequencing reads enabled efficient phasing of genetic variations, allowing the construction of genome-wide maps of phased SVs and SNVs. We employed read-based phasing to show that all de novo chromothripsis breakpoints occurred on paternal chromosomes and we resolved the long-range structure of the chromothripsis. This work demonstrates the value of long-read sequencing for screening whole genomes of patients for complex structural variants.

scholarly journals Improving the Chromosome-Level Genome Assembly of the Siamese Fighting Fish (Betta splendens) in a University Master’s Course

2020 ◽  
Vol 10 (7) ◽  
pp. 2179-2183 ◽  
Author(s):  
Stefan Prost ◽  
Malte Petersen ◽  
Martin Grethlein ◽  
Sarah Joy Hahn ◽  
Nina Kuschik-Maczollek ◽  
...  
Keyword(s):  
Genome Assembly ◽  
High Throughput Sequencing ◽  
Siamese Fighting Fish ◽  
Betta Splendens ◽  
High Quality ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Chromosome Level

Ever decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish (Betta splendens) that was carried out during a practical university master’s course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behavior. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published Hi-C data. The use of ∼35x nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using the Hi-C data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 96.1% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly. We present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university master’s course. The use of ∼35× long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.

scholarly journals Assessment of Bioleaching Microbial Community Structure and Function Based on Next-Generation Sequencing Technologies

Minerals ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 596 ◽  
Author(s):  
Shuang Zhou ◽  
Min Gan ◽  
Jianyu Zhu ◽  
Xinxing Liu ◽  
Guanzhou Qiu
Keyword(s):  
Community Structure ◽  
Next Generation Sequencing ◽  
Microbial Ecology ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Structure And Function ◽  
Next Generation ◽  
Sequencing Technologies ◽  
And Function ◽  
Generation Sequencing

It is widely known that bioleaching microorganisms have to cope with the complex extreme environment in which microbial ecology relating to community structure and function varies across environmental types. However, analyses of microbial ecology of bioleaching bacteria is still a challenge. To address this challenge, numerous technologies have been developed. In recent years, high-throughput sequencing technologies enabling comprehensive sequencing analysis of cellular RNA and DNA within the reach of most laboratories have been added to the toolbox of microbial ecology. The next-generation sequencing technology allowing processing DNA sequences can produce available draft genomic sequences of more bioleaching bacteria, which provides the opportunity to predict models of genetic and metabolic potential of bioleaching bacteria and ultimately deepens our understanding of bioleaching microorganism. High-throughput sequencing that focuses on targeted phylogenetic marker 16S rRNA has been effectively applied to characterize the community diversity in an ore leaching environment. RNA-seq, another application of high-throughput sequencing to profile RNA, can be for both mapping and quantifying transcriptome and has demonstrated a high efficiency in quantifying the changing expression level of each transcript under different conditions. It has been demonstrated as a powerful tool for dissecting the relationship between genotype and phenotype, leading to interpreting functional elements of the genome and revealing molecular mechanisms of adaption. This review aims to describe the high-throughput sequencing approach for bioleaching environmental microorganisms, particularly focusing on its application associated with challenges.

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

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
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 ◽  
Single Molecule Sequencing ◽  
Short Read ◽  
Hybrid Approaches ◽  
Long Read

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.

scholarly journals Improving the chromosome-level genome assembly of the Siamese fighting fish (Betta splendens) in a university Master’s course

2020 ◽  
Author(s):  
Stefan Prost ◽  
Malte Petersen ◽  
Martin Grethlein ◽  
Sarah Joy Hahn ◽  
Nina Kuschik-Maczollek ◽  
...  
Keyword(s):  
Genome Assembly ◽  
High Throughput Sequencing ◽  
Siamese Fighting Fish ◽  
Betta Splendens ◽  
High Quality ◽  
Sequencing Platform ◽  
Sequencing Technologies ◽  
Oxford Nanopore ◽  
Long Read ◽  
Chromosome Level

AbstractBackgroundEver decreasing costs along with advances in sequencing and library preparation technologies enable even small research groups to generate chromosome-level assemblies today. Here we report the generation of an improved chromosome-level assembly for the Siamese fighting fish (Betta splendens) that was carried out during a practical university Master’s course. The Siamese fighting fish is a popular aquarium fish and an emerging model species for research on aggressive behaviour. We updated the current genome assembly by generating a new long-read nanopore-based assembly with subsequent scaffolding to chromosome-level using previously published HiC data.FindingsThe use of nanopore-based long-read data sequenced on a MinION platform (Oxford Nanopore Technologies) allowed us to generate a baseline assembly of only 1,276 contigs with a contig N50 of 2.1 Mbp, and a total length of 441 Mbp. Scaffolding using previously published HiC data resulted in 109 scaffolds with a scaffold N50 of 20.7 Mbp. More than 99% of the assembly is comprised in 21 scaffolds. The assembly showed the presence of 95.8% complete BUSCO genes from the Actinopterygii dataset indicating a high quality of the assembly.ConclusionWe present an improved full chromosome-level assembly of the Siamese fighting fish generated during a university Master’s course. The use of ~35× long-read nanopore data drastically improved the baseline assembly in terms of continuity. We show that relatively in-expensive high-throughput sequencing technologies such as the long-read MinION sequencing platform can be used in educational settings allowing the students to gain practical skills in modern genomics and generate high quality results that benefit downstream research projects.

scholarly journals PgRC: Pseudogenome based Read Compressor

2019 ◽  
Author(s):  
Tomasz Kowalski ◽  
Szymon Grabowski
Keyword(s):  
High Throughput ◽  
Compression Ratio ◽  
High Throughput Sequencing ◽  
Sequencing Data ◽  
High Quality ◽  
Link Type ◽  
Sequencing Technologies ◽  
Significant Interest ◽  
The One ◽  
Shortest Common Superstring

AbstractMotivationThe amount of sequencing data from High-Throughput Sequencing technologies grows at a pace exceeding the one predicted by Moore’s law. One of the basic requirements is to efficiently store and transmit such huge collections of data. Despite significant interest in designing FASTQ compressors, they are still imperfect in terms of compression ratio or decompression resources.ResultsWe present Pseudogenome-based Read Compressor (PgRC), an in-memory algorithm for compressing the DNA stream, based on the idea of building an approximation of the shortest common superstring over high-quality reads. Experiments show that PgRC wins in compression ratio over its main competitors, SPRING and Minicom, by up to 18 and 21 percent on average, respectively, while being at least comparably fast in decompression.AvailabilityPgRC can be downloaded from https://github.com/kowallus/[email protected]

scholarly journals A Protocol for mtGenome Analysis on Large Sample Numbers

2014 ◽  
Vol 8 ◽  
pp. BBI.S14623 ◽  
Author(s):  
Igor G. Hamoy ◽  
André M. Ribeiro-Dos-Santos ◽  
Luiz Alvarez ◽  
Silvanira Barbosa ◽  
Artur Silva ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
High Throughput ◽  
Genome Analysis ◽  
High Throughput Sequencing ◽  
Complete Mitochondrial Genome ◽  
High Quality ◽  
Sequencing Technologies ◽  
Ion Pgm ◽  
Total Variability ◽  
Run Up

The mitochondrial genome is widely studied in a variety of fields, such as population, forensic, and human and medical genetics. Most studies have been limited to a small portion of the sequence that, although highly diverse, does not describe the total variability. The arrival of modern high-throughput sequencing technologies has made it possible to investigate larger sequences in a shorter amount of time as well as in a more affordable fashion. This work aims to describe a protocol for sequencing and analyzing the complete mitochondrial genome with the Ion PGM™ platform. To evaluate the protocol, the mitochondrial genome was sequenced to approximately 210 Mbp, with high-quality sequences distributed between 12 samples that had an average coverage of 1023× per sample. Several variant callers were compared to improve the protocol outcome. The results suggest that it is possible to run up to 120 samples per run without any loss of any significant quality. Therefore, this protocol is an efficient and accurate tool for full mitochondrial genome analysis.

scholarly journals Complete and high-quality genomes of novel microbial species from a meromictic lake using a workflow combining long- and short-read sequencing platforms

2021 ◽  
Author(s):  
Yu-Hsiang Chen ◽  
Pei-Wen Chiang ◽  
Denis Yu Rogozin ◽  
Andrey Georgievich Degermendzhy ◽  
Hsiu-Hui Chiu ◽  
...  
Keyword(s):  
Novel Species ◽  
Bacterial Genome ◽  
Meromictic Lake ◽  
High Quality ◽  
Good Opportunity ◽  
Short Read ◽  
Sequencing Technologies ◽  
Complete Genomes ◽  
Long Read ◽  
Sequencing Platforms

Background: Most of Earth's bacteria have yet to be cultivated. The metabolic and functional potentials of these uncultivated microorganisms thus remain mysterious, and the metagenome-assembled genome (MAG) approach is the most robust method for uncovering these potentials. However, MAGs discovered by conventional metagenomic assembly and binning methods are usually highly fragmented genomes with heterogeneous sequence contamination, and this affects the accuracy and sensitivity of genomic analyses. Though the maturation of long-read sequencing technologies provides a good opportunity to fix the problem of highly fragmented MAGs as mentioned above, the method's error-prone nature causes severe problems of long-read-alone metagenomics. Hence, methods are urgently needed to retrieve MAGs by a combination of both long- and short-read technologies to advance genome-centric metagenomics. Results: In this study, we combined Illumina and Nanopore data to develop a new workflow to reconstruct 233 MAGs-six novel bacterial orders, 20 families, 66 genera, and 154 species-from Lake Shunet, a secluded meromictic lake in Siberia. Those new MAGs were underrepresented or undetectable in other MAGs studies using metagenomes from human or other common organisms or habitats. Using this newly developed workflow and strategy, the average N50 of reconstructed MAGs greatly increased 10-40-fold compared to when the conventional Illumina assembly and binning method were used. More importantly, six complete MAGs were recovered from our datasets, five of which belong to novel species. We used these as examples to demonstrate many novel and intriguing genomic characteristics discovered in these newly complete genomes and proved the importance of high-quality complete MAGs in microbial genomics and metagenomics studies. Conclusions: The results show that it is feasible to apply our workflow with a few additional long reads to recover numerous complete and high-quality MAGs from short-read metagenomes of high microbial diversity environment samples. The unique features we identified from five complete genomes highlight the robustness of this method in genome-centric metagenomic research. The recovery of 154 novel species MAGs from a rarely explored lake greatly expands the current bacterial genome encyclopedia and broadens our knowledge by adding new genomic characteristics of bacteria. It demonstrates a strong need to recover MAGs from diverse unexplored habitats in the search for microbial dark matter.

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