De novo genome assembly and in natura epigenomics reveal salinity‐induced DNA methylation in the mangrove tree Bruguiera gymnorhiza

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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De Novo Transcriptome Assembly, Functional Annotation, and Transcriptome Dynamics Analyses Reveal Stress Tolerance Genes in Mangrove Tree (Bruguiera gymnorhiza)

International Journal of Molecular Sciences ◽

10.3390/ijms22189874 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9874

Author(s):

Matin Miryeganeh ◽

Hidetoshi Saze

Keyword(s):

Stress Tolerance ◽

Molecular Mechanisms ◽

High Salinity ◽

De Novo ◽

Transcriptome Assembly ◽

Expression Profiles ◽

Natural Habitats ◽

Mangrove Species ◽

Mangrove Tree ◽

Bruguiera Gymnorhiza

Their high adaptability to difficult coastal conditions makes mangrove trees a valuable resource and an interesting model system for understanding the molecular mechanisms underlying stress tolerance and adaptation of plants to the stressful environmental conditions. In this study, we used RNA sequencing (RNA-Seq) for de novo assembling and characterizing the Bruguiera gymnorhiza (L.) Lamk leaf transcriptome. B. gymnorhiza is one of the most widely distributed mangrove species from the biggest family of mangroves; Rhizophoraceae. The de novo assembly was followed by functional annotations and identification of individual transcripts and gene families that are involved in abiotic stress response. We then compared the genome-wide expression profiles between two populations of B. gymnorhiza, growing under different levels of stress, in their natural habitats. One population living in high salinity environment, in the shore of the Pacific Ocean- Japan, and the other population living about one kilometre farther from the ocean, and next to the estuary of a river; in less saline and more brackish condition. Many genes involved in response to salt and osmotic stress, showed elevated expression levels in trees growing next to the ocean in high salinity condition. Validation of these genes may contribute to future salt-resistance research in mangroves and other woody plants. Furthermore, the sequences and transcriptome data provided in this study are valuable scientific resources for future comparative transcriptome research in plants growing under stressful conditions.

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Accurate long-read de novo assembly evaluation with Inspector

Genome Biology ◽

10.1186/s13059-021-02527-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Yu Chen ◽

Yixin Zhang ◽

Amy Y. Wang ◽

Min Gao ◽

Zechen Chong

Keyword(s):

Genome Assembly ◽

De Novo Assembly ◽

In Silico ◽

Large Scale ◽

De Novo ◽

Small Scale ◽

De Novo Genome Assembly ◽

Consensus Sequences ◽

Assembly Evaluation ◽

Long Read

AbstractLong-read de novo genome assembly continues to advance rapidly. However, there is a lack of effective tools to accurately evaluate the assembly results, especially for structural errors. We present Inspector, a reference-free long-read de novo assembly evaluator which faithfully reports types of errors and their precise locations. Notably, Inspector can correct the assembly errors based on consensus sequences derived from raw reads covering erroneous regions. Based on in silico and long-read assembly results from multiple long-read data and assemblers, we demonstrate that in addition to providing generic metrics, Inspector can accurately identify both large-scale and small-scale assembly errors.

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De novo genome assembly and single nucleotide variations for Soybean yellow common mosaic virus using soybean flower bud transcriptome data

Journal of Applied Biological Chemistry ◽

10.3839/jabc.2020.026 ◽

2020 ◽

Vol 63 (3) ◽

pp. 189-195

Author(s):

Yeonhwa Jo ◽

Hoseong Choi ◽

Sang-Min Kim ◽

Bong Choon Lee ◽

Won Kyong Cho

Keyword(s):

Mosaic Virus ◽

Genome Assembly ◽

De Novo ◽

Transcriptome Data ◽

Flower Bud ◽

De Novo Genome Assembly ◽

Single Nucleotide ◽

Single Nucleotide Variations

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Gene Annotation and Transcriptome Delineation on a De Novo Genome Assembly for the Reference Leishmania major Friedlin Strain

Genes ◽

10.3390/genes12091359 ◽

2021 ◽

Vol 12 (9) ◽

pp. 1359

Author(s):

Esther Camacho ◽

Sandra González-de la Fuente ◽

Jose C. Solana ◽

Alberto Rastrojo ◽

Fernando Carrasco-Ramiro ◽

...

Keyword(s):

Genome Sequence ◽

Genome Assembly ◽

Molecular Mechanisms ◽

High Throughput Sequencing ◽

Leishmania Major ◽

De Novo ◽

Gene Annotation ◽

Leishmania Species ◽

De Novo Genome Assembly ◽

Sequencing Platforms

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.

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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.

Download Full-text