De novo assembly of highly polymorphic metagenomic data using in situ generated reference sequences and a novel BLAST-based assembly pipeline

Background. Metagenomics has transformed our understanding of microbial diversity across ecosystems, with recent advances enabling de novo assembly of genomes from metagenomes. These metagenome-assembled genomes are critical to provide ecological, evolutionary, and metabolic context for all the microbes and viruses yet to be cultivated. Metagenomes can now be generated from nanogram to subnanogram amounts of DNA. However, these libraries require several rounds of PCR amplification before sequencing, and recent data suggest these typically yield smaller and more fragmented assemblies than regular metagenomes. Methods. Here we evaluate de novo assembly methods of 169 PCR-amplified metagenomes, including 25 for which an unamplified counterpart is available, to optimize specific assembly approaches for PCR-amplified libraries. We first evaluated coverage bias by mapping reads from PCR-amplified metagenomes onto reference contigs obtained from unamplified metagenomes of the same samples. Then, we compared different assembly pipelines in terms of assembly size (number of bp in contigs ≥ 10kb) and error rates to evaluate which are the best suited for PCR-amplified metagenomes. Results. Read mapping analyses revealed that the depth of coverage within individual genomes is significantly more uneven in PCR-amplified datasets versus unamplified metagenomes, with regions of high depth of coverage enriched in short inserts. This enrichment scales with the number of PCR cycles performed, and is presumably due to preferential amplification of short inserts. Standard assembly pipelines are confounded by this type of coverage unevenness, so we evaluated other assembly options to mitigate these issues. We found that a pipeline combining read deduplication and an assembly algorithm originally designed to recover genomes from libraries generated after whole genome amplification (single-cell SPAdes) frequently improved assembly of contigs ≥ 10kb by 10 to 100-fold for low input metagenomes. Conclusions. PCR-amplified metagenomes have enabled scientists to explore communities traditionally challenging to describe, including some with extremely low biomass or from which DNA is particularly difficult to extract. Here we show that a modified assembly pipeline can lead to an improved de novo genome assembly from PCR-amplified datasets, and enables a better genome recovery from low input metagenomes.

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Reducing the number of artifactual repeats in de novo assembly of RNA-Seq data by optimizing the assembly pipeline

Gene Reports ◽

10.1016/j.genrep.2017.08.003 ◽

2017 ◽

Vol 9 ◽

pp. 7-12

Author(s):

Wei-Kang Lee ◽

Nur Afiza Mohd Zainuddin ◽

Hui-Ying Teh ◽

Yi-Yi Lim ◽

Mohd Uzair Jaafar ◽

...

Keyword(s):

De Novo Assembly ◽

De Novo ◽

Rna Seq ◽

Assembly Pipeline

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Telomere length de novo assembly of all 7 chromosomes and mitogenome sequencing of the model entomopathogenic fungus, Metarhizium brunneum, by means of a novel assembly pipeline

BMC Genomics ◽

10.1186/s12864-021-07390-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Zack Saud ◽

Alexandra M. Kortsinoglou ◽

Vassili N. Kouvelis ◽

Tariq M. Butt

Keyword(s):

De Novo Assembly ◽

De Novo ◽

Gene Prediction ◽

Fungal Species ◽

Orthologous Protein ◽

Metarhizium Brunneum ◽

Sequencing Technologies ◽

Protein Clusters ◽

Assembly Pipeline ◽

Generation Sequencing

Abstract Background More accurate and complete reference genomes have improved understanding of gene function, biology, and evolutionary mechanisms. Hybrid genome assembly approaches leverage benefits of both long, relatively error-prone reads from third-generation sequencing technologies and short, accurate reads from second-generation sequencing technologies, to produce more accurate and contiguous de novo genome assemblies in comparison to using either technology independently. In this study, we present a novel hybrid assembly pipeline that allowed for both mitogenome de novo assembly and telomere length de novo assembly of all 7 chromosomes of the model entomopathogenic fungus, Metarhizium brunneum. Results The improved assembly allowed for better ab initio gene prediction and a more BUSCO complete proteome set has been generated in comparison to the eight current NCBI reference Metarhizium spp. genomes. Remarkably, we note that including the mitogenome in ab initio gene prediction training improved overall gene prediction. The assembly was further validated by comparing contig assembly agreement across various assemblers, assessing the assembly performance of each tool. Genomic synteny and orthologous protein clusters were compared between Metarhizium brunneum and three other Hypocreales species with complete genomes, identifying core proteins, and listing orthologous protein clusters shared uniquely between the two entomopathogenic fungal species, so as to further facilitate the understanding of molecular mechanisms underpinning fungal-insect pathogenesis. Conclusions The novel assembly pipeline may be used for other haploid fungal species, facilitating the need to produce high-quality reference fungal genomes, leading to better understanding of fungal genomic evolution, chromosome structuring and gene regulation.

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Telomere length de novo assembly of all 7 chromosomes and mitogenome sequencing of the model entomopathogenic fungus, Metarhizium brunneum, by means of a novel assembly pipeline

10.21203/rs.3.rs-60098/v3 ◽

2020 ◽

Author(s):

Zack Saud ◽

Alexandra M. Kortsinoglou ◽

Vassili N. Kouvelis ◽

Tariq M. Butt

Keyword(s):

De Novo Assembly ◽

De Novo ◽

Gene Prediction ◽

Fungal Species ◽

Orthologous Protein ◽

Metarhizium Brunneum ◽

Sequencing Technologies ◽

Protein Clusters ◽

Assembly Pipeline ◽

Generation Sequencing

Abstract Background More accurate and complete reference genomes have improved understanding of gene function, biology, and evolutionary mechanisms. Hybrid genome assembly approaches leverage benefits of both long, relatively error-prone reads from third-generation sequencing technologies and short, accurate reads from second-generation sequencing technologies, to produce more accurate and contiguous de novo genome assemblies in comparison to using either technology independently. In this study, we present a novel hybrid assembly pipeline that allowed for both mitogenome de novo assembly and telomere length de novo assembly of all 7 chromosomes of the model entomopathogenic fungus, Metarhizium brunneum . Results The improved assembly allowed for better ab initio gene prediction and a more BUSCO complete proteome set has been generated in comparison to the eight current NCBI reference Metarhizium spp. genomes. Remarkably, we note that including the mitogenome in ab initio gene prediction training improved overall gene prediction. The assembly was further validated by comparing contig assembly agreement across various assemblers, assessing the assembly performance of each tool. Genomic synteny and orthologous protein clusters were compared between Metarhizium brunneum and three other Hypocreales species with complete genomes, identifying core proteins, and listing orthologous protein clusters shared uniquely between the two entomopathogenic fungal species, so as to further facilitate the understanding of molecular mechanisms underpinning fungal-insect pathogenesis. Conclusions The novel assembly pipeline may be used for other haploid fungal species, facilitating the need to produce high-quality reference fungal genomes, leading to better understanding of fungal genomic evolution, chromosome structuring and gene regulation.

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GPU acceleration of Darwin read overlapper for de novo assembly of long DNA reads

BMC Bioinformatics ◽

10.1186/s12859-020-03685-1 ◽

2020 ◽

Vol 21 (S13) ◽

Author(s):

Nauman Ahmed ◽

Tong Dong Qiu ◽

Koen Bertels ◽

Zaid Al-Ars

Keyword(s):

De Novo Assembly ◽

Large Scale ◽

De Novo ◽

State Of The Art ◽

Gpu Acceleration ◽

Dna Assembly ◽

Scoring Model ◽

Assembly Pipeline ◽

Scoring Schemes ◽

Gpu Implementation

Abstract Background In Overlap-Layout-Consensus (OLC) based de novo assembly, all reads must be compared with every other read to find overlaps. This makes the process rather slow and limits the practicality of using de novo assembly methods at a large scale in the field. Darwin is a fast and accurate read overlapper that can be used for de novo assembly of state-of-the-art third generation long DNA reads. Darwin is designed to be hardware-friendly and can be accelerated on specialized computer system hardware to achieve higher performance. Results This work accelerates Darwin on GPUs. Using real Pacbio data, our GPU implementation on Tesla K40 has shown a speedup of 109x vs 8 CPU threads of an Intel Xeon machine and 24x vs 64 threads of IBM Power8 machine. The GPU implementation supports both linear and affine gap, scoring model. The results show that the GPU implementation can achieve the same high speedup for different scoring schemes. Conclusions The GPU implementation proposed in this work shows significant improvement in performance compared to the CPU version, thereby making it accessible for utilization as a practical read overlapper in a DNA assembly pipeline. Furthermore, our GPU acceleration can also be used for performing fast Smith-Waterman alignment between long DNA reads. GPU hardware has become commonly available in the field today, making the proposed acceleration accessible to a larger public. The implementation is available at https://github.com/Tongdongq/darwin-gpu.

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Nanopore sequencing and de novo assembly of a misidentified Camelpox vaccine reveals putative epigenetic modifications and alternate protein signal peptides

Scientific Reports ◽

10.1038/s41598-021-97158-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zack Saud ◽

Matthew D. Hitchings ◽

Tariq M. Butt

Keyword(s):

Vaccinia Virus ◽

Virus Strain ◽

De Novo Assembly ◽

De Novo ◽

Epigenetic Modifications ◽

Signal Peptides ◽

Dna Viruses ◽

Sequencing Technologies ◽

Assembly Pipeline ◽

Vaccinia Virus Strain

AbstractDNA viruses can exploit host cellular epigenetic processes to their advantage; however, the epigenome status of most DNA viruses remains undetermined. Third generation sequencing technologies allow for the identification of modified nucleotides from sequencing experiments without specialized sample preparation, permitting the detection of non-canonical epigenetic modifications that may distinguish viral nucleic acid from that of their host, thus identifying attractive targets for advanced therapeutics and diagnostics. We present a novel nanopore de novo assembly pipeline used to assemble a misidentified Camelpox vaccine. Two confirmed deletions of this vaccine strain in comparison to the closely related Vaccinia virus strain modified vaccinia Ankara make it one of the smallest non-vector derived orthopoxvirus genomes to be reported. Annotation of the assembly revealed a previously unreported signal peptide at the start of protein A38 and several predicted signal peptides that were found to differ from those previously described. Putative epigenetic modifications around various motifs have been identified and the assembly confirmed previous work showing the vaccine genome to most closely resemble that of Vaccinia virus strain Modified Vaccinia Ankara. The pipeline may be used for other DNA viruses, increasing the understanding of DNA virus evolution, virulence, host preference, and epigenomics.

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PyDamage: automated ancient damage identification and estimation for contigs in ancient DNA de novo assembly

10.1101/2021.03.24.436838 ◽

2021 ◽

Author(s):

Maxime Borry ◽

Alexander Huebner ◽

Adam B Rohrlach ◽

Christina G Warinner

Keyword(s):

Dna Damage ◽

Functional Diversity ◽

Ancient Dna ◽

De Novo Assembly ◽

Damage Identification ◽

De Novo ◽

Metagenomic Data ◽

Cytosine Deamination ◽

High Data ◽

Gene Catalogue

DNA de novo assembly can be used to reconstruct longer stretches of DNA (contigs), including genes and even genomes, from short DNA sequencing reads. Applying this technique to metagenomic data derived from archaeological remains, such as paleofeces and dental calculus, we can investigate past microbiome functional diversity that may be absent or underrepresented in the modern microbiome gene catalogue. However, compared to modern samples, ancient samples are often burdened with environmental contamination, resulting in metagenomic datasets that represent mixtures of ancient and modern DNA. The ability to rapidly and reliably establish the authenticity and integrity of ancient samples is essential for ancient DNA studies, and the ability to distinguish between ancient and modern sequences is particularly important for ancient microbiome studies. Characteristic patterns of ancient DNA damage, namely DNA fragmentation and cytosine deamination (observed as C-to-T transitions) are typically used to authenticate ancient samples and sequences. However, existing tools for inspecting and filtering aDNA damage either compute it at the read level, which leads to high data loss and lower quality when used in combination with de novo assembly, or require manual inspection, which is impractical for ancient assemblies that typically contain tens to hundreds of thousands of contigs. To address these challenges, we designed PyDamage, a robust, automated approach for aDNA damage estimation and authentication of de novo assembled aDNA. PyDamage uses a likelihood ratio based approach to discriminate between truly ancient contigs and contigs originating from modern contamination. We test PyDamage on both simulated, and empirical aDNA data from archaeological paleofeces, and we demonstrate its ability to reliably and automatically identify contigs bearing DNA damage characteristic of aDNA. Coupled with aDNA de novo assembly, PyDamage opens up new doors to explore functional diversity in ancient metagenomic datasets.

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Ultra-deep, long-read nanopore sequencing of mock microbial community standards

10.1101/487033 ◽

2018 ◽

Cited By ~ 5

Author(s):

Samuel M Nicholls ◽

Joshua C Quick ◽

Shuiquan Tang ◽

Nicholas J Loman

Keyword(s):

Microbial Community ◽

De Novo Assembly ◽

De Novo ◽

Limit Of Detection ◽

Read Length ◽

Metagenomic Data ◽

Mock Community ◽

Microbial Species ◽

Long Read ◽

Community Standards

Background: Long sequencing reads are information-rich: aiding de novo assembly and reference mapping, and consequently have great potential for the study of microbial communities. However, the best approaches for analysis of long-read metagenomic data are unknown. Additionally, rigorous evaluation of bioinformatics tools is hindered by a lack of long-read data from validated samples with known composition. Methods: We sequenced two commercially-available mock communities containing ten microbial species (ZymoBIOMICS Microbial Community Standards) with Oxford Nanopore GridION and PromethION. Isolates from the same mock community were sequenced individually with Illumina HiSeq. Data: We generated 14 and 16 Gbp from GridION flowcells and 146 and 148 Gbp from PromethION flowcells for the even and odd communities respectively. Read length N50 was 5.3 Kbp and 5.2 Kbp for the even and log community, respectively. Basecalls and corresponding signal data are made available (4.2 TB in total). Results: Alignment to Illumina-sequenced isolates demonstrated the expected microbial species at anticipated abundances, with the limit of detection for the lowest abundance species below 50 cells (GridION). De novo assembly of metagenomes recovered long contiguous sequences without the need for pre-processing techniques such as binning. Conclusions: We present ultra-deep, long-read nanopore datasets from a well-defined mock community. These datasets will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and comparison of current laboratory and software pipelines.

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Semi-Automatic In Silico Gap Closure Enabled De Novo Assembly of Two Dehalobacter Genomes from Metagenomic Data

PLoS ONE ◽

10.1371/journal.pone.0052038 ◽

2012 ◽

Vol 7 (12) ◽

pp. e52038 ◽

Cited By ~ 35

Author(s):

Shuiquan Tang ◽

Yunchen Gong ◽

Elizabeth A. Edwards

Keyword(s):

De Novo Assembly ◽

In Silico ◽

De Novo ◽

Metagenomic Data ◽

Gap Closure

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Optimizing de novo genome assembly from PCR-amplified metagenomes

10.7287/peerj.preprints.27453v1 ◽

2018 ◽

Author(s):

Simon Roux ◽

Gareth Trubl ◽

Danielle Goudeau ◽

Nandita Nath ◽

Estelle Couradeau ◽

...

Keyword(s):

Genome Assembly ◽

De Novo Assembly ◽

De Novo ◽

Pcr Amplification ◽

Error Rates ◽

De Novo Genome Assembly ◽

Low Input ◽

Assembly Algorithm ◽

Coverage Bias ◽

Assembly Pipeline

Background. Metagenomics has transformed our understanding of microbial diversity across ecosystems, with recent advances enabling de novo assembly of genomes from metagenomes. These metagenome-assembled genomes are critical to provide ecological, evolutionary, and metabolic context for all the microbes and viruses yet to be cultivated. Metagenomes can now be generated from nanogram to subnanogram amounts of DNA. However, these libraries require several rounds of PCR amplification before sequencing, and recent data suggest these typically yield smaller and more fragmented assemblies than regular metagenomes. Methods. Here we evaluate de novo assembly methods of 169 PCR-amplified metagenomes, including 25 for which an unamplified counterpart is available, to optimize specific assembly approaches for PCR-amplified libraries. We first evaluated coverage bias by mapping reads from PCR-amplified metagenomes onto reference contigs obtained from unamplified metagenomes of the same samples. Then, we compared different assembly pipelines in terms of assembly size (number of bp in contigs ≥ 10kb) and error rates to evaluate which are the best suited for PCR-amplified metagenomes. Results. Read mapping analyses revealed that the depth of coverage within individual genomes is significantly more uneven in PCR-amplified datasets versus unamplified metagenomes, with regions of high depth of coverage enriched in short inserts. This enrichment scales with the number of PCR cycles performed, and is presumably due to preferential amplification of short inserts. Standard assembly pipelines are confounded by this type of coverage unevenness, so we evaluated other assembly options to mitigate these issues. We found that a pipeline combining read deduplication and an assembly algorithm originally designed to recover genomes from libraries generated after whole genome amplification (single-cell SPAdes) frequently improved assembly of contigs ≥ 10kb by 10 to 100-fold for low input metagenomes. Conclusions. PCR-amplified metagenomes have enabled scientists to explore communities traditionally challenging to describe, including some with extremely low biomass or from which DNA is particularly difficult to extract. Here we show that a modified assembly pipeline can lead to an improved de novo genome assembly from PCR-amplified datasets, and enables a better genome recovery from low input metagenomes.

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