Long-read metagenomics of multiple displacement amplified DNA of low-biomass human gut phageomes by SACRA preprocessing chimeric reads

Abstract The human gut bacteriophage community (phageome) plays an important role in the host’s health and disease; however, the entire structure is poorly understood, partly owing to the generation of many incomplete genomes in conventional short-read metagenomics. Here, we show long-read metagenomics of amplified DNA of low-biomass phageomes with multiple displacement amplification (MDA), involving the development of a novel bioinformatics tool, SACRA, that efficiently preprocessed numerous chimeric reads generated through MDA. Using five samples, SACRA markedly reduced the average chimera ratio from 72% to 1.5% in PacBio reads with an average length of 1.8-kb. De novo assembly of chimera-less PacBio long reads reconstructed contigs of ≥ 5-kb with an average proportion of 27%, which was 1% in contigs from MiSeq short reads, thereby dramatically improving contig length and genome completeness. Comparison of PacBio and MiSeq contigs found MiSeq contig fragmentations frequently near local repeats and hypervariable regions in the phage genomes, and those caused by multiple homologous phage genomes coexisting in the community. We also developed a reference-independent method to assess the completeness of the linear phage genomes. Overall, we established a SACRA-coupled long-read metagenomics robust to highly diverse gut phageomes, identifying high-quality circular and linear phage genomes with adequate sequence quantity.

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

10.21203/rs.3.rs-58640/v1 ◽

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.

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Long-read whole genome analysis of human single cells

10.1101/2021.04.13.439527 ◽

2021 ◽

Author(s):

Joanna Hård ◽

Jeff E Mold ◽

Jesper Eisfeldt ◽

Christian Tellgren-Roth ◽

Susana Häggqvist ◽

...

Keyword(s):

Genetic Variation ◽

Single Cell ◽

De Novo ◽

Multiple Displacement Amplification ◽

Single Cells ◽

Whole Genome Analysis ◽

Human Donor ◽

Amplification Bias ◽

Long Reads ◽

Long Read

With long-read sequencing we have entered an era where individual genomes are routinely assembled to near-completion and where complex genetic variation can efficiently be resolved. Here we demonstrate that long reads can be applied also to study the genomic architecture of individual human cells. Clonally expanded CD8+ T-cells from a human donor were used as starting material for a droplet-based multiple displacement amplification (dMDA) method designed to ensure long molecule lengths and minimal amplification bias. Sequencing of two single cells was performed on the PacBio Sequel II system, generating over 2.5 million reads and ~20Gb HiFi data (>QV20) per cell, achieving up to 40% genome coverage. This data allowed for single nucleotide variant (SNV) detection, including in genomic regions inaccessible by short reads. Over 1000 high-confidence structural variants (SVs) per cell were discovered in the PacBio data, which is four times more than the number of SVs detected in Illumina dMDA data from clonally related cells. In addition, several putative clone-specific somatic SV events could be identified. Single-cell de novo assembly resulted in 454-598 Mb assembly sizes and 35-42 kb contig N50 values. 1762 (12.8%) of expected gene models were found to be complete in the best single-cell assembly. The de novo constructed mitochondrial genomes were 100% identical for the two single cells subjected to PacBio sequencing, although mitochondrial heteroplasmy was also observed. In summary, the work presented here demonstrates the utility of long-read sequencing towards understanding the extent and distribution of complex genetic variation at the single cell level.

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A previously undescribed highly prevalent phage identified in a Danish enteric virome catalogue

10.21203/rs.3.rs-273865/v1 ◽

2021 ◽

Author(s):

Lore Van Espen ◽

Emilie Glad Bak ◽

Leen Beller ◽

Lila Close ◽

Ward Deboutte ◽

...

Keyword(s):

Viral Genome ◽

De Novo ◽

Human Gut ◽

Host Genus ◽

Faecal Samples ◽

High Prevalence ◽

Paediatric Cohort ◽

Health And Disease ◽

Wet Lab ◽

Public Datasets

Abstract Background: Gut viruses are important players in the complex human gut microbial ecosystem. Recently, the number of human gut virome studies is steadily increasing, however we are still only scratching the surface of the immense viral diversity as many wet lab and bio-informatics challenges remain. In this study, 254 virus-enriched faecal metagenomes from 204 Danish subjects were used to generate a Danish Enteric Virome Catalogue (DEVoC) of 12,986 non-redundant viral genome sequences encoding 190,029 viral genes, which formed 67,921 orthologous groups. The DEVoC was used to characterize the composition of the healthy DEVoC gut viromes from 46 children and adolescents (6-18 years old) and 45 adults (40 -73 years old).Results: The majority of DEVoC viral sequences (67.3 %) and proteins (61.6 %) were not present in other (human gut) viral genome databases. Gut viromes of healthy Danish subjects mostly consisted of phages. While 39 phage genomes (PGs) were present in more than 10 healthy subjects, the degree of viral individuality was high. Among the 39 prevalent PGs, one was significantly more prevalent in the paediatric cohort, whereas two were more prevalent in adults. In 1,880 gut virome samples of 27 studies from across the world, the 39 prevalent PGs reveal several age-, geography- and disease-related prevalence patterns. Two PGs also showed a remarkably high prevalence worldwide – a crAss-like phage (20.6% prevalence), belonging to the tentative AlphacrAssvirinae subfamily, genus I; and a previously undescribed circular temperate phage (14.4% prevalence), named LoVEphage (because it encodes Lots of Viral Elements). A de novo assembly of selected public datasets generated an additional 18 circular LoVEphage-like genomes (67.9-72.4 kb). CRISPR spacer analysis suggested Bacteroides as a host genus for the LoVEphage, and a closely related prophage was identified in Bacteroides dorei, further confirming the host.Conclusions: The DEVoC, the largest human gut virome catalogue generated from consistently processed faecal samples, facilitated analysis of healthy Danish human gut viromes and we foresee that it will benefit future analysis on the roles of gut viruses in human health and disease. The identification of a previously undescribed prevalent phage illustrates the usefulness of developing a virome catalogue.

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Platanus_B: an accurate de novo assembler for bacterial genomes using an iterative error-removal process

DNA Research ◽

10.1093/dnares/dsaa014 ◽

2020 ◽

Vol 27 (3) ◽

Cited By ~ 1

Author(s):

Rei Kajitani ◽

Dai Yoshimura ◽

Yoshitoshi Ogura ◽

Yasuhiro Gotoh ◽

Tetsuya Hayashi ◽

...

Keyword(s):

High Resolution ◽

Large Scale ◽

De Novo ◽

Bacterial Genomes ◽

Long Reads ◽

Wide Range ◽

Long Read ◽

Phylogenomic Analyses ◽

Error Removal ◽

Iterative Error

Abstract De novo assembly of short DNA reads remains an essential technology, especially for large-scale projects and high-resolution variant analyses in epidemiology. However, the existing tools often lack sufficient accuracy required to compare closely related strains. To facilitate such studies on bacterial genomes, we developed Platanus_B, a de novo assembler that employs iterations of multiple error-removal algorithms. The benchmarks demonstrated the superior accuracy and high contiguity of Platanus_B, in addition to its ability to enhance the hybrid assembly of both short and nanopore long reads. Although the hybrid strategies for short and long reads were effective in achieving near full-length genomes, we found that short-read-only assemblies generated with Platanus_B were sufficient to obtain ≥90% of exact coding sequences in most cases. In addition, while nanopore long-read-only assemblies lacked fine-scale accuracies, inclusion of short reads was effective in improving the accuracies. Platanus_B can, therefore, be used for comprehensive genomic surveillances of bacterial pathogens and high-resolution phylogenomic analyses of a wide range of bacteria.

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

10.1101/2020.12.22.423164 ◽

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.

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Fast-SG: An alignment-free algorithm for hybrid assembly

10.1101/209122 ◽

2017 ◽

Author(s):

Alex Di Genova ◽

Gonzalo A. Ruz ◽

Marie-France Sagot ◽

Alejandro Maass

Keyword(s):

De Novo ◽

Reference Level ◽

Hybrid Assembly ◽

Short Read ◽

Sequencing Technologies ◽

Alignment Free ◽

Long Reads ◽

Long Read ◽

Definition Of ◽

Large Genomes

ABSTRACTLong read sequencing technologies are the ultimate solution for genome repeats, allowing near reference level reconstructions of large genomes. However, long read de novo assembly pipelines are computationally intense and require a considerable amount of coverage, thereby hindering their broad application to the assembly of large genomes. Alternatively, hybrid assembly methods which combine short and long read sequencing technologies can reduce the time and cost required to produce de novo assemblies of large genomes. In this paper, we propose a new method, called FAST-SG, which uses a new ultra-fast alignment-free algorithm specifically designed for constructing a scaffolding graph using light-weight data structures. FAST-SG can construct the graph from either short or long reads. This allows the reuse of efficient algorithms designed for short read data and permits the definition of novel modular hybrid assembly pipelines. Using comprehensive standard datasets and benchmarks, we show how FAST-SG outperforms the state-of-the-art short read aligners when building the scaffolding graph, and can be used to extract linking information from either raw or error-corrected long reads. We also show how a hybrid assembly approach using FAST-SG with shallow long read coverage (5X) and moderate computational resources can produce long-range and accurate reconstructions of the genomes of Arabidopsis thaliana (Ler-0) and human (NA12878).

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De novo genome assembly of the olive fruit fly (Bactrocera oleae) developed through a combination of linked-reads and long-read technologies

10.1101/505040 ◽

2018 ◽

Cited By ~ 1

Author(s):

Haig Djambazian ◽

Anthony Bayega ◽

Konstantina T. Tsoumani ◽

Efthimia Sagri ◽

Maria-Eleni Gregoriou ◽

...

Keyword(s):

Y Chromosome ◽

De Novo ◽

Fruit Fly ◽

Bactrocera Oleae ◽

Olive Fruit Fly ◽

Olive Fruit ◽

Long Reads ◽

Oxford Nanopore ◽

Long Read ◽

Oxford Nanopore Technologies

AbstractLong-read sequencing has greatly contributed to the generation of high quality assemblies, albeit at a high cost. It is also not always clear how to combine sequencing platforms. We sequenced the genome of the olive fruit fly (Bactrocera oleae), the most important pest in the olive fruits agribusiness industry, using Illumina short-reads, mate-pairs, 10x Genomics linked-reads, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT). The 10x linked-reads assembly gave the most contiguous assembly with an N50 of 2.16 Mb. Scaffolding the linked-reads assembly using long-reads from ONT gave a more contiguous assembly with scaffold N50 of 4.59 Mb. We also present the most extensive transcriptome datasets of the olive fly derived from different tissues and stages of development. Finally, we used the Chromosome Quotient method to identify Y-chromosome scaffolds and show that the long-reads based assembly generates very highly contiguous Y-chromosome assembly.JR is a member of the MinION Access Program (MAP) and has received free-of-charge flow cells and sequencing kits from Oxford Nanopore Technologies for other projects. JR has had no other financial support from ONT.AB has received re-imbursement for travel costs associated with attending Nanopore Community meeting 2018, a meeting organized my Oxford Nanopore Technologies.

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The evaluation of RNA-Seq de novo assembly by PacBio long read sequencing

10.1101/735621 ◽

2019 ◽

Author(s):

Yifan Yang ◽

Michael Gribskov

Keyword(s):

Real Time ◽

De Novo ◽

Critical Issue ◽

Evaluation Methods ◽

Model Organisms ◽

Rna Seq ◽

Long Reads ◽

Long Read ◽

Set Up ◽

Downstream Analysis

AbstractRNA-Seq de novo assembly is an important method to generate transcriptomes for non-model organisms before any downstream analysis. Given many great de novo assembly methods developed by now, one critical issue is that there is no consensus on the evaluation of de novo assembly methods yet. Therefore, to set up a benchmark for evaluating the quality of de novo assemblies is very critical. Addressing this challenge will help us deepen the insights on the properties of different de novo assemblers and their evaluation methods, and provide hints on choosing the best assembly sets as transcriptomes of non-model organisms for the further functional analysis. In this article, we generate a “real time” transcriptome using PacBio long reads as a benchmark for evaluating five de novo assemblers and two model-based de novo assembly evaluation methods. By comparing the de novo assmblies generated by RNA-Seq short reads with the “real time” transcriptome from the same biological sample, we find that Trinity is best at the completeness by generating more assemblies than the alternative assemblers, but less continuous and having more misassemblies; Oases is best at the continuity and specificity, but less complete; The performance of SOAPdenovo-Trans, Trans-AByss and IDBA-Tran are in between of five assemblers. For evaluation methods, DETONATE leverages multiple aspects of the assembly set and ranks the assembly set with an average performance as the best, meanwhile the contig score can serve as a good metric to select assemblies with high completeness, specificity, continuity but not sensitive to misassemblies; TransRate contig score is useful for removing misassemblies, yet often the assemblies in the optimal set is too few to be used as a transcriptome.

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

10.1101/2019.12.20.884486 ◽

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.

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De novo clustering of long-read transcriptome data using a greedy, quality-value based algorithm

10.1101/463463 ◽

2018 ◽

Cited By ~ 8

Author(s):

Kristoffer Sahlin ◽

Paul Medvedev

Keyword(s):

Clustering Algorithm ◽

De Novo ◽

Substantial Improvement ◽

Error Rates ◽

Reconstruction Algorithms ◽

Long Reads ◽

Oxford Nanopore ◽

Long Read ◽

Transcript Reconstruction ◽

Oxford Nanopore Technologies

AbstractLong-read sequencing of transcripts with PacBio Iso-Seq and Oxford Nanopore Technologies has proven to be central to the study of complex isoform landscapes in many organisms. However, current de novo transcript reconstruction algorithms from long-read data are limited, leaving the potential of these technologies unfulfilled. A common bottleneck is the dearth of scalable and accurate algorithms for clustering long reads according to their gene family of origin. To address this challenge, we develop isONclust, a clustering algorithm that is greedy (in order to scale) and makes use of quality values (in order to handle variable error rates). We test isONclust on three simulated and five biological datasets, across a breadth of organisms, technologies, and read depths. Our results demonstrate that isONclust is a substantial improvement over previous approaches, both in terms of overall accuracy and/or scalability to large datasets. Our tool is available at https://github.com/ksahlin/isONclust.

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