Whole-genome Shotgun Approach Research Articles

A whole-genome shotgun approach to human reference genome sequencing.

A whole-genome shotgun approach to human reference genome sequencing.

Development and Validation of a Real-Time PCR for the Molecular Identification of Thaumatotibia leucotreta (Lepidoptera: Tortricidae: Olethreutinae) Intercepted in Trade

The false codling moth, Thaumatotibia leucotreta (Meyrick), is a polyphagous pest indigenous to most of sub-Saharan Africa. This species is considered to be quarantined throughout much of the world, in part due to its extensive host range and significant damage caused by larval feeding. We developed a specific real-time PCR assay that allows for rapid and reliable identification of T. leucotreta. More than 150 target specimens were sequenced using an Illumina whole-genome shotgun approach to identify the most suitable loci for assay development. A hydrolysis probe that binds to a segment of the internal transcribed spacer 2 (ITS2) region was designed and used with a generic internal control targeting 18S rDNA. The assay was examined for cross-reactivity by testing additional Thaumatotibia species and representatives of related Olethreutine leafroller genera such as Cryptophlebia, Cydia, and Grapholita, which are often encountered in the same geographic region and on the same hosts as T. leucotreta. We compared our newly developed test to previously published TaqMan real-time PCR, SYBR Green real-time PCR, and loop-mediated isothermal amplification (LAMP) tests. Our newly developed real-time PCR assay outperformed all three tests in terms of analytical specificity, with 100% accuracy. These results will help to further improve diagnostic standards for molecular identification of T. leucotreta. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Metagenomic Approach to Bacterial Diversity and Lipolytic Enzymes’ Genes from a Steam Soil of Los Humeros Geothermal Field (Puebla, México)

Los Humeros volcanic complex, located at the eastern end of the Trans Mexican Volcanic Belt, possess superficial thermal manifestations (fumaroles, heat steam-soils and alteration zones), with temperatures between 50 °C and 90 °C. This is the first work to explore the bacterial diversity of a steam soil of Los Humeros geothermal field using a metagenomic 16S RNA gene analysis, and the presence of lipolytic enzymes’ genes after an enrichment culture by a whole-genome shotgun approach. High diversity indexes were found in soil samples, with Firmicutes, Chloroflexi, Proteobacteria, Actinobacteria and Acidobacteria as the most prevalent phyla. Alicyclobacillus was the most prevalent genus at 15 cm and Anoxybacillus at 50 cm depth; anaerobic and obligate thermophilic and hyperthermophilic members were found at 50 cm depth. Firmicutes was favored by the oil-rich medium, and the dominant genera were Geobacillus and Caldibacillus. 165 genes related to lipolytic enzymes were found; carboxilesterases and phospholipases C were the most abundant, and 43% were assigned to uncharacterized lipolytic enzymes. The diversity as well as the high percentage of non-characterized lipolytic enzymes found after soil enrichment in an oil-rich medium suggest the presence of biotechnologically promising organisms and enzymes that deserve to be investigated.

Shotgun metagenomic data of microbiomes on plastic fabrics exposed to harsh tropical environments.

The development of more affordable high-throughput DNA sequencing technologies and powerful bioinformatics is making of shotgun metagenomics a common tool for effective characterization of microbiomes and robust functional genomics. A shotgun metagenomic approach was applied in the characterization of microbial communities associated with plasticized fabric materials exposed to a harsh tropical environment for 14 months. High-throughput sequencing of TruSeq paired-end libraries was conducted using a whole-genome shotgun (WGS) approach on an Illumina HiSeq2000 platform generating 100 bp reads. A multifaceted bioinformatics pipeline was developed and applied to conduct quality control and trimming of raw reads, microbial classification, assembly of multi-microbial genomes, binning of assembled contigs to individual genomes, and prediction of microbial genes and proteins. The bioinformatic analysis of the large 161 Gb sequence dataset generated 3,314,688 contigs and 120 microbial genomes. The raw metagenomic data and the detailed description of the bioinformatics pipeline applied in data analysis provide an important resource for the genomic characterization of microbial communities associated with biodegraded plastic fabric materials. The raw shotgun metagenomics sequence data of microbial communities on plastic fabric materials have been deposited in MG-RAST (https://www.mg-rast.org/) under accession numbers: mgm4794685.3–mgm4794690.3. The datasets and raw data presented here were associated with the main research work “Metagenomic characterization of microbial communities on plasticized fabric materials exposed to harsh tropical environments” (Radwan et al., 2020).

Shotgun Metagenomic Sequencing Reveals Functional Genes and Microbiome Associated with Bovine Digital Dermatitis

Metagenomic methods amplifying 16S ribosomal RNA genes have been used to describe the microbial diversity of healthy skin and lesion stages of bovine digital dermatitis (DD) and to detect critical pathogens involved with disease pathogenesis. In this study, we characterized the microbiome and for the first time, the composition of functional genes of healthy skin (HS), active (ADD) and inactive (IDD) lesion stages using a whole-genome shotgun approach. Metagenomic sequences were annotated using MG-RAST pipeline. Six phyla were identified as the most abundant. Firmicutes and Actinobacteria were the predominant bacterial phyla in the microbiome of HS, while Spirochetes, Bacteroidetes and Proteobacteria were highly abundant in ADD and IDD. T. denticola-like, T. vincentii-like and T. phagedenis-like constituted the most abundant species in ADD and IDD. Recruitment plots comparing sequences from HS, ADD and IDD samples to the genomes of specific Treponema spp., supported the presence of T. denticola and T. vincentii in ADD and IDD. Comparison of the functional composition of HS to ADD and IDD identified a significant difference in genes associated with motility/chemotaxis and iron acquisition/metabolism. We also provide evidence that the microbiome of ADD and IDD compared to that of HS had significantly higher abundance of genes associated with resistance to copper and zinc, which are commonly used in footbaths to prevent and control DD. In conclusion, the results from this study provide new insights into the HS, ADD and IDD microbiomes, improve our understanding of the disease pathogenesis and generate unprecedented knowledge regarding the functional genetic composition of the digital dermatitis microbiome.

Decoding the oak genome: public release of sequence data, assembly, annotation and publication strategies.

The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole-genome shotgun (WGS) approach, without the use of costly and time-consuming methods, such as fosmid or BAC clone-based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS-FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired-end and mate-pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired-end reads and contaminants detected, resulting in a total of 17,910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome.

Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0582-8) contains supplementary material, which is available to authorized users.

Comparative BAC‐based physical mapping of Oryza sativa ssp. indica var. 93–11 and evaluation of the two rice reference sequence assemblies

Reference sequences are sequences that are used for public consultation, and therefore must be of high quality. Using the whole-genome shotgun/next-generation sequencing approach, many genome sequences of complex higher plants have been generated in recent years, and are generally considered reference sequences. However, none of these sequences has been experimentally evaluated at the whole-genome sequence assembly level. Rice has a relatively simple plant genome, and the genome sequences for its two sub-species obtained using different sequencing approaches were published approximately 10years ago. This provides a unique system for a case study to evaluate the qualities and utilities of published plant genome sequences. We constructed a robust BAC physical map embedding a large number of BAC end sequences forrice variety 93-11. Through BAC end sequence alignments and tri-assembly comparisons of the 93-11 physical map and the two reference sequences, we found that the Nipponbare reference sequence generated using the clone-by-clone approach has a high quality but still contains small artifact inversions and missing sequences. In contrast, the 93-11 reference sequence generated using the whole-genome shotgun approach contains many large and varied assembly errors, such as inversions, duplications and translocations, as well as missing sequences. The 93-11 physical map provides an invaluable resource for evaluation and improvements toward completion of both Nipponbare and 93-11 reference sequences.

Complete nucleotide sequence of pHN7A8, an F33:A-:B-type epidemic plasmid carrying blaCTX-M-65, fosA3 and rmtB from China

To characterize a representative self-transmissible multidrug resistance plasmid pHN7A8 isolated from an Escherichia coli from a dog in China, classified as F33:A-:B- by replicon sequence typing and carrying the bla(TEM-1b), bla(CTX-M-65), fosA3 and rmtB genes conferring resistance to penicillins, cephalosporins, fosfomycin and aminoglycosides, respectively. pHN7A8 was sequenced using a whole-genome shotgun approach and the sequence analysed by comparison with reference plasmids. pHN7A8 is a circular molecule of 76 878 bp. bla(CTX-M-65), fosA3 and rmtB are found in known contexts, interspersed with different mobile elements including ISEcp1, IS1, Tn2, IS1294, IS903 and four copies of IS26. This multiresistance region has only a single nucleotide difference from that of pXZ, an F2:A-:B- plasmid isolated from poultry in China. The pHN7A8 backbone carries genes encoding addiction and partitioning systems that promote plasmid maintenance and has a similar organization to pXZ, as well as IncFII plasmids such as R100, pC15-1a/pEK516 and pHK23, isolated in Japan, Canada/the UK and China, respectively, but with varying levels of identity, suggesting recombination. pHN7A8 is a chimera that may have resulted from the acquisition, by recombination in the plasmid backbone, of the multiresistance region found in pXZ. This region appears to have evolved from the resistance determinant R100 through the stepwise integration of multiple antimicrobial resistance determinants from different sources by the actions of mobile elements and recombination. The successful dissemination of this multidrug resistance plasmid presents further challenges for the prevention and treatment of Enterobacteriaceae infections.

Draft Genome Sequence of Lactobacillus rossiae DSM 15814 T

The draft genome sequence of Lactobacillus rossiae DSM 15814(T) (CS1, ATCC BAA-88) was determined by a whole-genome shotgun approach. Reads were assembled to a 2.9-Mb draft version. RAST genome annotation evidenced 2,723 predicted coding sequences. Many carbohydrate, amino acid, and amino acid derivative subsystem features were found.

Novel members of the phosphate regulon in Escherichia coli O157:H7 identified using a whole-genome shotgun approach

Escherichia coli PhoB protein is the transcriptional activator of the phosphate (pho) regulon genes involved in phosphate utilization. To gain further insight into the potential roles of PhoB in the phosphate starvation response, we attempted to identify PhoB-regulated promoters using a random shotgun library of E. coli O157:H7 genomic fragments that were fused to a promoterless lacZ reporter gene on a low-copy-number plasmid. Using this approach, numerous chromosomal regions containing phosphate-starvation-inducible (psi) promoters, including nearly all known pho regulon promoters, were identified. β-Galactosidase and electrophoretic mobility shift assays showed that transcription from the 22 identified psi promoters was directly regulated by PhoB. PhoB-binding sites within the promoter regions were identified by DNase I footprinting. The genes for yoaI, rpsG, galP, rnr, udp, sstT, ybiM, and vgrE were located downstream of these promoters, indicating that these genes are members of the pho regulon. Surprisingly, the other 14 promoters were located within sense or antisense strands of open reading frames (ORFs), and/or at a distance from ORFs. Our results suggest that PhoB has broader roles in gene regulation and RNA expression in E. coli strains than was previously supposed. Our shotgun-library cloning approach represents a powerful tool for identifying promoters activated or repressed by transcriptional regulators that respond to environmental stimuli.

Characterization of the rumen microbiota of pre‐ruminant calves using metagenomic tools

The temporal sequence of microbial establishment in the rumen of the neonatal ruminant has important ecological and pathophysiological implications. In this study, we characterized the rumen microbiota of pre-ruminant calves fed milk replacer using two approaches, pyrosequencing of hypervariable V3-V5 regions of the 16S rRNA gene and whole-genome shotgun approach. Fifteen bacterial phyla were identified in the microbiota of pre-ruminant calves. Bacteroidetes was the predominant phylum in the rumen microbiota of 42-day-old calves, representing 74.8% of the 16S sequences, followed by Firmicutes (12.0%), Proteobacteria (10.4%), Verrucomicrobia (1.2%) and Synergistetes (1.1%). However, the phylum-level composition of 14-day-old calves was distinctly different. A total of 170 bacterial genera were identified while the core microbiome of pre-ruminant calves included 45 genera. Rumen development seemingly had a significant impact on microbial diversity. The dazzling functional diversity of the rumen microbiota was reflected by identification of 8298 Pfam and 3670 COG protein families. The rumen microbiota of pre-ruminant calves displayed a considerable compositional heterogeneity during early development. This is evidenced by a profound difference in rumen microbial composition between the two age groups. However, all functional classes between the two age groups had a remarkably similar assignment, suggesting that rumen microbial communities of pre-ruminant calves maintained a stable function and metabolic potentials while their phylogenetic composition fluctuated greatly. The presence of all major types of rumen microorganisms suggests that the rumen of pre-ruminant calves may not be rudimentary. Our results provide insight into rumen microbiota dynamics and will facilitate efforts in formulating optimal early-weaning strategies.

Metagenomic Insights into the Evolution, Function, and Complexity of the Planktonic Microbial Community of Lake Lanier, a Temperate Freshwater Ecosystem

Lake Lanier is an important freshwater lake for the southeast United States, as it represents the main source of drinking water for the Atlanta metropolitan area and is popular for recreational activities. Temperate freshwater lakes such as Lake Lanier are underrepresented among the growing number of environmental metagenomic data sets, and little is known about how functional gene content in freshwater communities relates to that of other ecosystems. To better characterize the gene content and variability of this freshwater planktonic microbial community, we sequenced several samples obtained around a strong summer storm event and during the fall water mixing using a random whole-genome shotgun (WGS) approach. Comparative metagenomics revealed that the gene content was relatively stable over time and more related to that of another freshwater lake and the surface ocean than to soil. However, the phylogenetic diversity of Lake Lanier communities was distinct from that of soil and marine communities. We identified several important genomic adaptations that account for these findings, such as the use of potassium (as opposed to sodium) osmoregulators by freshwater organisms and differences in the community average genome size. We show that the lake community is predominantly composed of sequence-discrete populations and describe a simple method to assess community complexity based on population richness and evenness and to determine the sequencing effort required to cover diversity in a sample. This study provides the first comprehensive analysis of the genetic diversity and metabolic potential of a temperate planktonic freshwater community and advances approaches for comparative metagenomics.

Micro‐collinearity and genome evolution in the vicinity of an ethylene receptor gene of cultivated diploid and allotetraploid coffee species (Coffea)

Arabica coffee (Coffea arabica L.) is a self-compatible perennial allotetraploid species (2n=4x=44), whereas Robusta coffee (C. canephora L.) is a self-incompatible perennial diploid species (2n=2x=22). C. arabica (C(a) C(a) E(a) E(a) ) is derived from a spontaneous hybridization between two closely related diploid coffee species, C. canephora (CC) and C. eugenioides (EE). To investigate the patterns and degree of DNA sequence divergence between the Arabica and Robusta coffee genomes, we identified orthologous bacterial artificial chromosomes (BACs) from C. arabica and C. canephora, and compared their sequences to trace their evolutionary history. Although a high level of sequence similarity was found between BACs from C. arabica and C. canephora, numerous chromosomal rearrangements were detected, including inversions, deletions and insertions. DNA sequence identity between C. arabica and C. canephora orthologous BACs ranged from 93.4% (between E(a) and C(a) ) to 94.6% (between C(a) and C). Analysis of eight orthologous gene pairs resulted in estimated ages of divergence between 0.046 and 0.665 million years, indicating a recent origin of the allotetraploid species C. arabica. Analysis of transposable elements revealed differential insertion events that contributed to the size increase in the C(a) sub-genome compared to its diploid relative. In particular, we showed that insertion of a Ty1-copia LTR retrotransposon occurred specifically in C. arabica, probably shortly after allopolyploid formation. The two sub-genomes of C. arabica, C(a) and E(a) , showed sufficient sequence differences, and a whole-genome shotgun approach could be suitable for sequencing the allotetraploid genome of C. arabica.

The Pinus taeda genome is characterized by diverse and highly diverged repetitive sequences.

BackgroundIn today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24). The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda.ResultsWe present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS) sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (≥ 75% nucleotide identity) elsewhere in the genome, but only 23% have identical copies (99% identity). The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome.ConclusionsThis study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is a feasible goal.

Genome sequence of the palaeopolyploid soybean

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create a chromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70% more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78% of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies. The two duplication events were followed by gene diversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.

Complete nucleotide sequence of pKP96, a 67 850 bp multiresistance plasmid encoding qnrA1, aac(6′)-Ib-cr and blaCTX-M-24 from Klebsiella pneumoniae

The multiresistance plasmid pKP96 from Klebsiella pneumoniae was sequenced completely and analysed concerning its genetic environment and distributing of antimicrobial resistance genes. The complete sequence of the plasmid was determined using a whole-genome shotgun approach. MICs of 13 antimicrobial agents were determined using Etests. A conjugation experiment was performed in liquid medium. pKP96 is a circularly closed 67 850 bp multiresistance plasmid with an IncN incompatibility group. Seventy putative genes were identified according to the annotation of the finished sequence. The backbone region of the plasmid, comprising the conjugal transfer and plasmid replication regions, showed 91% identity to the IncN plasmid R46. Several mobile elements were found to be inserted into pKP96 together with antimicrobial resistance genes, including qnrA1, aac(6')-Ib-cr and bla(CTX-M-24). Plasmid pKP96 is a chimera that has acquired its multiple antimicrobial resistance determinants horizontally from different sources. It may have evolved from an ancestor plasmid similar to R46 through the stepwise events of integration or recombination.

2× genomes—Does depth matter?: Table 1.

2× genomes—Does depth matter?: Table 1.

Complete Nucleotide Sequence of pK245, a 98-Kilobase Plasmid Conferring Quinolone Resistance and Extended-Spectrum-β-Lactamase Activity in a Clinical Klebsiella pneumoniae Isolate

A plasmid containing the qnrS quinolone resistance determinant and the gene encoding the SHV-2 beta-lactamase has been discovered from a clinical Klebsiella pneumoniae strain isolated in Taiwan. The complete 98-kb sequence of this plasmid, designated pK245, was determined by using a whole-genome shotgun approach. Transfer of pK245 conferred low-level resistance to fluoroquinolones in electroporant Escherichia coli epi300. The sequence of the immediate region surrounding qnrS in pK245 is nearly identical (>99% identity) to those of pAH0376 from Shigella flexneri and pINF5 from Salmonella enterica serovar Infantis, the two other qnrS-carrying plasmids reported to date, indicating a potential common origin. Other genes conferring resistance to aminoglycosides (aacC2, strA, and strB), chloramphenicol (catA2), sulfonamides (sul2), tetracycline (tetD), and trimethoprim (dfrA14) were also detected in pK245. The dfrA14 gene is carried on a class I integron. Several features of this plasmid, including three separate regions containing putative replicons, a partitioning-control system, and a type II restriction modification system, suggest that it may be able to replicate and adapt in a variety of hosts. Although no critical conjugative genes were detected, multiple insertion sequence elements were found scattered throughout pK245, and these may facilitate the dissemination of the antimicrobial resistance determinants. We conclude that pK245 is a chimera which acquired its multiple antimicrobial resistance determinants horizontally from different sources. The identification of pK245 plasmid expands the repertoire of the coexistence of quinolone and extended-spectrum-beta-lactam resistance determinants in plasmids carried by various species of the family Enterobacteriaceae in different countries.

Fly's time

The struggle between public and private efforts to sequence the fly genome is the subject of Michael Ashburner's new book, Won for All: How the Drosophila Genome Was Sequenced