A high-quality de novo genome assembly from a single parasitoid wasp

AbstractSequencing and assembling a genome with a single individual have several advantages, such as lower heterozygosity and easier sample preparation. However, the amount of genomic DNA of some small sized organisms might not meet the standard DNA input requirement for current sequencing pipelines. Although few studies sequenced a single small insect with about 100 ng DNA as input, it may still be challenging for many small organisms to obtain such amount of DNA from a single individual. Here, we use 20 ng DNA as input, and present a high-quality genome assembly for a single haploid male parasitoid wasp (Habrobracon hebetor) using Nanopore and Illumina. Because of the low input DNA, a whole genome amplification (WGA) method is used before sequencing. The assembled genome size is 131.6 Mb with a contig N50 of 1.63 Mb. A total of 99% Benchmarking Universal Single-Copy Orthologs are detected, suggesting the high level of completeness of the genome assembly. Genome comparison between H. hebetor and its relative Bracon brevicornis shows a high-level genome synteny, indicating the genome of H. hebetor is highly accurate and contiguous. Our study provides an example for de novo assembling a genome from ultra-low input DNA, and will be used for sequencing projects of small sized species and rare samples, haploid genomics as well as population genetics of small sized species.

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Chromatin Architectures Are Associated with Response to Dark Treatment in the Oil Crop Sesamum indicum, Based on a High-Quality Genome Assembly

Plant and Cell Physiology ◽

10.1093/pcp/pcaa026 ◽

2020 ◽

Vol 61 (5) ◽

pp. 978-987

Author(s):

Chaoqiong Li ◽

Xiaoli Li ◽

Hongzhan Liu ◽

Xueqin Wang ◽

Weifeng Li ◽

...

Keyword(s):

Gene Transcription ◽

Genome Assembly ◽

De Novo ◽

Hierarchical Structures ◽

Normal Growth ◽

High Quality ◽

Dark Treatment ◽

De Novo Genome Assembly ◽

Chromosome Conformation ◽

A Genome

Abstract Eukaryotic chromatin is tightly packed into hierarchical structures, allowing appropriate gene transcription in response to environmental and developmental cues. Here, we provide a chromosome-scale de novo genome assembly of sesame with a total length of 292.3 Mb and a scaffold N50 of 20.5 Mb, containing estimated 28,406 coding genes using Pacific Biosciences long reads combined with a genome-wide chromosome conformation capture (Hi-C) approach. Based on this high-quality reference genome, we detected changes in chromatin architectures between normal growth and dark-treated sesame seedlings. Gene expression level was significantly higher in ‘A’ compartment and topologically associated domain (TAD) boundary regions than in ‘B’ compartment and TAD interior regions, which is coincident with the enrichment of H4K3me3 modification in these regions. Moreover, differentially expressed genes (DEGs) induced by dark treated were enriched in the changed TAD-related regions and genomic differential contact regions. Gene Ontology (GO) enrichment analysis of DEGs showed that genes related to ‘response to stress’ and ‘photosynthesis’ functional categories were enriched, which corresponds to dark treatment. These results suggested that chromatin organization is associated with gene transcription in response to dark treatment in sesame. Our results will facilitate the understanding of regulatory mechanisms in response to environmental cues in plants.

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Optimizing de novo genome assembly from PCR-amplified metagenomes

PeerJ ◽

10.7717/peerj.6902 ◽

2019 ◽

Vol 7 ◽

pp. e6902 ◽

Cited By ~ 9

Author(s):

Simon Roux ◽

Gareth Trubl ◽

Danielle Goudeau ◽

Nandita Nath ◽

Estelle Couradeau ◽

...

Keyword(s):

Genome Assembly ◽

De Novo ◽

Pcr Amplification ◽

Error Rates ◽

De Novo Genome Assembly ◽

Low Input ◽

Assembly Algorithm ◽

Coverage Bias ◽

Size Number ◽

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 ≥ 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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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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Genome assembly of the JD17 soybean provides a new reference genome for Comparative genomics

10.1101/2021.11.23.469778 ◽

2021 ◽

Author(s):

Xinxin Yi ◽

Jing Liu ◽

Shengcai Chen ◽

Hao Wu ◽

Min Liu ◽

...

Keyword(s):

Nitrogen Fixation ◽

Genome Assembly ◽

Reference Genome ◽

De Novo ◽

Genomic Analysis ◽

Comparative Genomic ◽

High Quality ◽

Genome Wide ◽

A Genome ◽

Cultivated Soybean

Cultivated soybean (Glycine max) is an important source for protein and oil. Many elite cultivars with different traits have been developed for different conditions. Each soybean strain has its own genetic diversity, and the availability of more high-quality soybean genomes can enhance comparative genomic analysis for identifying genetic underpinnings for its unique traits. In this study, we constructed a high-quality de novo assembly of an elite soybean cultivar Jidou 17 (JD17) with chromsome contiguity and high accuracy. We annotated 52,840 gene models and reconstructed 74,054 high-quality full-length transcripts. We performed a genome-wide comparative analysis based on the reference genome of JD17 with three published soybeans (WM82, ZH13 and W05) , which identified five large inversions and two large translocations specific to JD17, 20,984 - 46,912 PAVs spanning 13.1 - 46.9 Mb in size, and 5 - 53 large PAV clusters larger than 500kb. 1,695,741 - 3,664,629 SNPs and 446,689 - 800,489 Indels were identified and annotated between JD17 and them. Symbiotic nitrogen fixation (SNF) genes were identified and the effects from these variants were further evaluated. It was found that the coding sequences of 9 nitrogen fixation-related genes were greatly affected. The high-quality genome assembly of JD17 can serve as a valuable reference for soybean functional genomics research.

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A high-quality genome assembly from a single, field-collected spotted lanternfly (Lycorma delicatula) using the PacBio Sequel II system

GigaScience ◽

10.1093/gigascience/giz122 ◽

2019 ◽

Vol 8 (10) ◽

Cited By ~ 12

Author(s):

Sarah B Kingan ◽

Julie Urban ◽

Christine C Lambert ◽

Primo Baybayan ◽

Anna K Childers ◽

...

Keyword(s):

Invasive Species ◽

Genome Assembly ◽

De Novo ◽

Fragment Size ◽

High Quality ◽

De Novo Genome Assembly ◽

Lycorma Delicatula ◽

Long Read ◽

Genome Assemblies ◽

High Quality Genome

ABSTRACT Background A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies; however, long-read methods have historically had greater input DNA requirements and higher costs than next-generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female spotted lanternfly (Lycorma delicatula) using a single Pacific Biosciences SMRT Cell. The spotted lanternfly is an invasive species recently discovered in the northeastern United States that threatens to damage economically important crop plants in the region. Results The DNA from 1 individual was used to make 1 standard, size-selected library with an average DNA fragment size of ∼20 kb. The library was run on 1 Sequel II SMRT Cell 8M, generating a total of 132 Gb of long-read sequences, of which 82 Gb were from unique library molecules, representing ∼36× coverage of the genome. The assembly had high contiguity (contig N50 length = 1.5 Mb), completeness, and sequence level accuracy as estimated by conserved gene set analysis (96.8% of conserved genes both complete and without frame shift errors). Furthermore, it was possible to segregate more than half of the diploid genome into the 2 separate haplotypes. The assembly also recovered 2 microbial symbiont genomes known to be associated with L. delicatula, each microbial genome being assembled into a single contig. Conclusions We demonstrate that field-collected arthropods can be used for the rapid generation of high-quality genome assemblies, an attractive approach for projects on emerging invasive species, disease vectors, or conservation efforts of endangered species.

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A high-quality, long-read de novo genome assembly to aid conservation of Hawaii’s last remaining crow species

10.1101/349035 ◽

2018 ◽

Author(s):

Jolene T. Sutton ◽

Martin Helmkampf ◽

Cynthia C. Steiner ◽

M. Renee Bellinger ◽

Jonas Korlach ◽

...

Keyword(s):

Genome Assembly ◽

Captive Breeding ◽

De Novo ◽

Bird Species ◽

Population Level ◽

Model Systems ◽

Population Declines ◽

High Quality ◽

De Novo Genome Assembly ◽

Long Read

AbstractGenome-level data can provide researchers with unprecedented precision to examine the causes and genetic consequences of population declines, and to apply these results to conservation management. Here we present a high-quality, long-read, de novo genome assembly for one of the world’s most endangered bird species, the Alala. As the only remaining native crow species in Hawaii, the Alala survived solely in a captive breeding program from 2002 until 2016, at which point a long-term reintroduction program was initiated. The high-quality genome assembly was generated to lay the foundation for both comparative genomics studies, and the development of population-level genomic tools that will aid conservation and recovery efforts. We illustrate how the quality of this assembly places it amongst the very best avian genomes assembled to date, comparable to intensively studied model systems. We describe the genome architecture in terms of repetitive elements and runs of homozygosity, and we show that compared with more outbred species, the Alala genome is substantially more homozygous. We also provide annotations for a subset of immunity genes that are likely to be important for conservation applications, and we discuss how this genome is currently being used as a roadmap for downstream conservation applications.

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A near complete genome for goat genetic and genomic research

Genetics Selection Evolution ◽

10.1186/s12711-021-00668-5 ◽

2021 ◽

Vol 53 (1) ◽

Author(s):

Ran Li ◽

Peng Yang ◽

Xuelei Dai ◽

Hojjat Asadollahpour Nanaei ◽

Wenwen Fang ◽

...

Keyword(s):

Y Chromosome ◽

Genome Assembly ◽

De Novo ◽

Genetic Research ◽

Genomic Research ◽

Dairy Goat ◽

De Novo Genome Assembly ◽

Important Species ◽

Long Read ◽

High Level

Abstract Background Goat, one of the first domesticated livestock, is a worldwide important species both culturally and economically. The current goat reference genome, known as ARS1, is reported as the first nonhuman genome assembly using 69× PacBio sequencing. However, ARS1 suffers from incomplete X chromosome and highly fragmented Y chromosome scaffolds. Results Here, we present a very high-quality de novo genome assembly, Saanen_v1, from a male Saanen dairy goat, with the first goat Y chromosome scaffold based on 117× PacBio long-read sequencing and 118× Hi-C data. Saanen_v1 displays a high level of completeness thanks to the presence of centromeric and telomeric repeats at the proximal and distal ends of two-thirds of the autosomes, and a much reduced number of gaps (169 vs. 773). The completeness and accuracy of the Saanen_v1 genome assembly are also evidenced by more assembled sequences on the chromosomes (2.63 Gb for Saanen_v1 vs. 2.58 Gb for ARS1), a slightly increased mapping ratio for transcriptomic data, and more genes anchored to chromosomes. The eight putative large assembly errors (1 to ~ 7 Mb each) found in ARS1 were amended, and for the first time, the substitution rate of this ruminant Y chromosome was estimated. Furthermore, sequence improvement in Saanen_v1, compared with ARS1, enables us to assign the likely correct positions for 4.4% of the single nucleotide polymorphism (SNP) probes in the widely used GoatSNP50 chip. Conclusions The updated goat genome assembly including both sex chromosomes (X and Y) and the autosomes with high-resolution quality will serve as a valuable resource for goat genetic research and applications.

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High-Quality de novo Genome Assembly of Huajingxian 74, a Receptor Parent of Single Segment Substitution Lines

Rice Science ◽

10.1016/j.rsci.2020.09.010 ◽

2021 ◽

Vol 28 (2) ◽

pp. 109-113

Author(s):

Li Fangping ◽

Gao Yanhao ◽

Wu Bingqi ◽

Cai Qingpei ◽

Zhan Pengling ◽

...

Keyword(s):

Genome Assembly ◽

De Novo ◽

Substitution Lines ◽

High Quality ◽

De Novo Genome Assembly ◽

Single Segment ◽

Single Segment Substitution Lines ◽

Segment Substitution

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LongStitch: High-quality genome assembly correction and scaffolding using long reads

10.1101/2021.06.17.448848 ◽

2021 ◽

Author(s):

Lauren Coombe ◽

Janet X Li ◽

Theodora Lo ◽

Johnathan Wong ◽

Vladimir Nikolic ◽

...

Keyword(s):

Genome Assembly ◽

De Novo ◽

Draft Genome ◽

Model Organisms ◽

High Quality ◽

De Novo Genome Assembly ◽

Long Reads ◽

Long Read ◽

Genomic Regions ◽

Genome Assemblies

Background Generating high-quality de novo genome assemblies is foundational to the genomics study of model and non-model organisms. In recent years, long-read sequencing has greatly benefited genome assembly and scaffolding, a process by which assembled sequences are ordered and oriented through the use of long-range information. Long reads are better able to span repetitive genomic regions compared to short reads, and thus have tremendous utility for resolving problematic regions and helping generate more complete draft assemblies. Here, we present LongStitch, a scalable pipeline that corrects and scaffolds draft genome assemblies exclusively using long reads. Results LongStitch incorporates multiple tools developed by our group and runs in up to three stages, which includes initial assembly correction (Tigmint-long), followed by two incremental scaffolding stages (ntLink and ARKS-long). Tigmint-long and ARKS-long are misassembly correction and scaffolding utilities, respectively, previously developed for linked reads, that we adapted for long reads. Here, we describe the LongStitch pipeline and introduce our new long-read scaffolder, ntLink, which utilizes lightweight minimizer mappings to join contigs. LongStitch was tested on short and long-read assemblies of three different human individuals using corresponding nanopore long-read data, and improves the contiguity of each assembly from 2.0-fold up to 304.6-fold (as measured by NGA50 length). Furthermore, LongStitch generates more contiguous and correct assemblies compared to state-of-the-art long-read scaffolder LRScaf in most tests, and consistently runs in under five hours using less than 23GB of RAM. Conclusions Due to its effectiveness and efficiency in improving draft assemblies using long reads, we expect LongStitch to benefit a wide variety of de novo genome assembly projects. The LongStitch pipeline is freely available at https://github.com/bcgsc/longstitch.

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A High-Quality De Novo Genome Assembly from a Single Mosquito using PacBio Sequencing

10.1101/499954 ◽

2018 ◽

Author(s):

Sarah B. Kingan ◽

Haynes Heaton ◽

Juliana Cudini ◽

Christine C. Lambert ◽

Primo Baybayan ◽

...

Keyword(s):

Single Molecule ◽

Genome Assembly ◽

Population Genomics ◽

De Novo ◽

Anopheles Coluzzii ◽

High Quality ◽

De Novo Genome Assembly ◽

Core Technology ◽

Conserved Genes ◽

Diploid Individual

AbstractA high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (∼5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 hour movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes are present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes are present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

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