Integrative Meta-Assembly Pipeline (IMAP): Chromosome-level genome assembler combining multiple de novo assemblies

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 ≥ 10 kb) 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 ≥10 kb 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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Chromosome-level assembly of Drosophila bifasciata reveals important karyotypic transition of the X chromosome

10.1101/847558 ◽

2019 ◽

Author(s):

Ryan Bracewell ◽

Anita Tran ◽

Kamalakar Chatla ◽

Doris Bachtrog

Keyword(s):

X Chromosome ◽

Genome Assembly ◽

De Novo ◽

Pericentromeric Region ◽

Species Group ◽

Chromosome 15 ◽

Protein Coding ◽

Protein Coding Genes ◽

Long Read ◽

Chromosome Level

ABSTRACTThe Drosophila obscura species group is one of the most studied clades of Drosophila and harbors multiple distinct karyotypes. Here we present a de novo genome assembly and annotation of D. bifasciata, a species which represents an important subgroup for which no high-quality chromosome-level genome assembly currently exists. We combined long-read sequencing (Nanopore) and Hi-C scaffolding to achieve a highly contiguous genome assembly approximately 193Mb in size, with repetitive elements constituting 30.1% of the total length. Drosophila bifasciata harbors four large metacentric chromosomes and the small dot, and our assembly contains each chromosome in a single scaffold, including the highly repetitive pericentromere, which were largely composed of Jockey and Gypsy transposable elements. We annotated a total of 12,821 protein-coding genes and comparisons of synteny with D. athabasca orthologs show that the large metacentric pericentromeric regions of multiple chromosomes are conserved between these species. Importantly, Muller A (X chromosome) was found to be metacentric in D. bifasciata and the pericentromeric region appears homologous to the pericentromeric region of the fused Muller A-AD (XL and XR) of pseudoobscura/affinis subgroup species. Our finding suggests a metacentric ancestral X fused to a telocentric Muller D and created the large neo-X (Muller A-AD) chromosome ∼15 MYA. We also confirm the fusion of Muller C and D in D. bifasciata and show that it likely involved a centromere-centromere fusion.

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A practical guide to buildde-novoassemblies for single tissues of non-model organisms: the example of a Neotropical frog

PeerJ ◽

10.7717/peerj.3702 ◽

2017 ◽

Vol 5 ◽

pp. e3702 ◽

Cited By ~ 5

Author(s):

Santiago Montero-Mendieta ◽

Manfred Grabherr ◽

Henrik Lantz ◽

Ignacio De la Riva ◽

Jennifer A. Leonard ◽

...

Keyword(s):

Defense Mechanisms ◽

De Novo ◽

Transcriptome Assembly ◽

Cost Effective ◽

Model Organisms ◽

Rna Seq ◽

Assembly Pipeline ◽

Wide Variability ◽

History Of ◽

Inexperienced User

Whole genome sequencing (WGS) is a very valuable resource to understand the evolutionary history of poorly known species. However, in organisms with large genomes, as most amphibians, WGS is still excessively challenging and transcriptome sequencing (RNA-seq) represents a cost-effective tool to explore genome-wide variability. Non-model organisms do not usually have a reference genome and the transcriptome must be assembledde-novo. We used RNA-seq to obtain the transcriptomic profile forOreobates cruralis, a poorly known South American direct-developing frog. In total, 550,871 transcripts were assembled, corresponding to 422,999 putative genes. Of those, we identified 23,500, 37,349, 38,120 and 45,885 genes present in the Pfam, EggNOG, KEGG and GO databases, respectively. Interestingly, our results suggested that genes related to immune system and defense mechanisms are abundant in the transcriptome ofO. cruralis. We also present a pipeline to assist with pre-processing, assembling, evaluating and functionally annotating ade-novotranscriptome from RNA-seq data of non-model organisms. Our pipeline guides the inexperienced user in an intuitive way through all the necessary steps to buildde-novotranscriptome assemblies using readily available software and is freely available at:https://github.com/biomendi/TRANSCRIPTOME-ASSEMBLY-PIPELINE/wiki.

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Optimizing de novo genome assembly from PCR-amplified metagenomes

10.7287/peerj.preprints.27453 ◽

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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De novo chromosome-level genome assembly of Chinese walnut (Juglans cathayensis Dode)

10.22541/au.159007642.20504888 ◽

2020 ◽

Author(s):

Feng Yan ◽

Rui Min Xi ◽

Rui Xue She ◽

Yu Jie Yan ◽

Peng Peng Chen ◽

...

Keyword(s):

Genome Assembly ◽

De Novo ◽

Chinese Walnut ◽

Chromosome Level

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Chromosome-level de novo genome assembly of Sarcophaga peregrina provides insights into the evolutionary adaptation of flesh flies

Authorea ◽

10.22541/au.158446791.17846463 ◽

2020 ◽

Author(s):

Lipin Ren ◽

Yanjie Shang ◽

Li Yang ◽

Shiwen Wang ◽

Xiang Wang ◽

...

Keyword(s):

Genome Assembly ◽

De Novo ◽

Evolutionary Adaptation ◽

De Novo Genome Assembly ◽

Flesh Flies ◽

Chromosome Level

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De Novo Assembly of a High-Quality Reference Genome for the Horned Lark (Eremophila alpestris)

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400846 ◽

2019 ◽

Vol 10 (2) ◽

pp. 475-478 ◽

Cited By ~ 3

Author(s):

Nicholas A. Mason ◽

Paulo Pulgarin ◽

Carlos Daniel Cadena ◽

Irby J. Lovette

Keyword(s):

Genome Assembly ◽

De Novo ◽

Single Copy ◽

Single Copy Gene ◽

High Quality ◽

Data Set ◽

Copy Gene ◽

Assembly Pipeline ◽

Horned Lark ◽

Gene Orthologs

The Horned Lark (Eremophila alpestris) is a small songbird that exhibits remarkable geographic variation in appearance and habitat across an expansive distribution. While E. alpestris has been the focus of many ecological and evolutionary studies, we still lack a highly contiguous genome assembly for the Horned Lark and related taxa (Alaudidae). Here, we present CLO_EAlp_1.0, a highly contiguous assembly for E. alpestris generated from a blood sample of a wild, male bird captured in the Altiplano Cundiboyacense of Colombia. By combining short-insert and mate-pair libraries with the ALLPATHS-LG genome assembly pipeline, we generated a 1.04 Gb assembly comprised of 2713 scaffolds, with a largest scaffold size of 31.81 Mb, a scaffold N50 of 9.42 Mb, and a scaffold L50 of 30. These scaffolds were assembled from 23685 contigs, with a largest contig size of 1.69 Mb, a contig N50 of 193.81 kb, and a contig L50 of 1429. Our assembly pipeline also produced a single mitochondrial DNA contig of 14.00 kb. After polishing the genome, we identified 94.5% of single-copy gene orthologs from an Aves data set and 97.7% of single-copy gene orthologs from a vertebrata data set, which further demonstrates the high quality of our assembly. We anticipate that this genomic resource will be useful to the broader ornithological community and those interested in studying the evolutionary history and ecological interactions of larks, which comprise a widespread, yet understudied lineage of songbirds.

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The sequencing and de novo assembly of the Larimichthys crocea genome using PacBio and Hi-C technologies

Scientific Data ◽

10.1038/s41597-019-0194-3 ◽

2019 ◽

Vol 6 (1) ◽

Cited By ~ 8

Author(s):

Baohua Chen ◽

Zhixiong Zhou ◽

Qiaozhen Ke ◽

Yidi Wu ◽

Huaqiang Bai ◽

...

Keyword(s):

Marine Fish ◽

Single Molecule ◽

Large Scale ◽

Reference Genome ◽

De Novo ◽

Larimichthys Crocea ◽

Chromosome Conformation ◽

Protein Coding ◽

Total Length ◽

Chromosome Level

Abstract Larimichthys crocea is an endemic marine fish in East Asia that belongs to Sciaenidae in Perciformes. L. crocea has now been recognized as an “iconic” marine fish species in China because not only is it a popular food fish in China, it is a representative victim of overfishing and still provides high value fish products supported by the modern large-scale mariculture industry. Here, we report a chromosome-level reference genome of L. crocea generated by employing the PacBio single molecule sequencing technique (SMRT) and high-throughput chromosome conformation capture (Hi-C) technologies. The genome sequences were assembled into 1,591 contigs with a total length of 723.86 Mb and a contig N50 length of 2.83 Mb. After chromosome-level scaffolding, 24 scaffolds were constructed with a total length of 668.67 Mb (92.48% of the total length). Genome annotation identified 23,657 protein-coding genes and 7262 ncRNAs. This highly accurate, chromosome-level reference genome of L. crocea provides an essential genome resource to support the development of genome-scale selective breeding and restocking strategies of L. crocea.

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Chromosome-Level Assembly of Drosophila bifasciata Reveals Important Karyotypic Transition of the X Chromosome

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400922 ◽

2020 ◽

Vol 10 (3) ◽

pp. 891-897 ◽

Cited By ~ 3

Author(s):

Ryan Bracewell ◽

Anita Tran ◽

Kamalakar Chatla ◽

Doris Bachtrog

Keyword(s):

X Chromosome ◽

Genome Assembly ◽

De Novo ◽

Pericentromeric Region ◽

Species Group ◽

Chromosome 15 ◽

Protein Coding ◽

Protein Coding Genes ◽

Long Read ◽

Chromosome Level

The Drosophila obscura species group is one of the most studied clades of Drosophila and harbors multiple distinct karyotypes. Here we present a de novo genome assembly and annotation of D. bifasciata, a species which represents an important subgroup for which no high-quality chromosome-level genome assembly currently exists. We combined long-read sequencing (Nanopore) and Hi-C scaffolding to achieve a highly contiguous genome assembly approximately 193 Mb in size, with repetitive elements constituting 30.1% of the total length. Drosophila bifasciata harbors four large metacentric chromosomes and the small dot, and our assembly contains each chromosome in a single scaffold, including the highly repetitive pericentromeres, which were largely composed of Jockey and Gypsy transposable elements. We annotated a total of 12,821 protein-coding genes and comparisons of synteny with D. athabasca orthologs show that the large metacentric pericentromeric regions of multiple chromosomes are conserved between these species. Importantly, Muller A (X chromosome) was found to be metacentric in D. bifasciata and the pericentromeric region appears homologous to the pericentromeric region of the fused Muller A-AD (XL and XR) of pseudoobscura/affinis subgroup species. Our finding suggests a metacentric ancestral X fused to a telocentric Muller D and created the large neo-X (Muller A-AD) chromosome ∼15 MYA. We also confirm the fusion of Muller C and D in D. bifasciata and show that it likely involved a centromere-centromere fusion.

Download Full-text