Genome Assembly Methods Research Articles

GTasm: a genome assembly method using graph transformers and HiFi reads

MotivationGenome assembly aims to reconstruct the whole chromosome-scale genome sequence. Obtaining accurate and complete chromosome-scale genome sequence serve as an indispensable foundation for downstream genomics analyses. Due to the complex repeat regions contained in genome sequence, the assembly results commonly are fragmented. Long reads with high accuracy rate can greatly enhance the integrity of genome assembly results.ResultsHere we introduce GTasm, an assembly method that uses graph transformer network to find optimal assembly results based on assembly graphs. Based on assembly graph, GTasm first extracts features about vertices and edges. Then, GTasm scores the edges by graph transformer model, and adopt a heuristic algorithm to find optimal paths in the assembly graph, each path corresponding to a contig. The graph transformer model is trained using simulated HiFi reads from CHM13, and GTasm is compared with other assembly methods using real HIFI read set. Through experimental result, GTasm can produce well assembly results, and achieve good performance on NA50 and NGA50 evaluation indicators. Applying deep learning models to genome assembly can improve the continuity and accuracy of assembly results. The code is available from https://github.com/chu-xuezhe/GTasm.

Three decades of rat genomics: approaching the finish(ed) line.

The rat, Rattus norvegicus, has provided an important model for investigation of a range of characteristics of biomedical importance. Here we survey the origins of this species, its introduction into laboratory research and the emergence of genetic and genomic methods that utilize this model organism. Genomic studies have yielded important progress and provided new insight into several biologically important traits. However, some studies have been impeded by the lack of a complete and accurate reference genome for this species. New sequencing and genome assembly methods applied to the rat have resulted in a new reference genome assembly, GRCr8, which is a near telomere-to-telomere assembly of high base level accuracy that incorporates several elements not captured in prior assemblies. As genome assembly methods continue to advance and production costs become a less significant obstacle, genome assemblies for multiple inbred rat strains are emerging. These assemblies will allow a rat pangenome assembly to be constructed which captures all the genetic variation in strains selected for their utility in research and will overcome reference bias, a limitation associated with reliance on a single reference assembly. By this means, the full utility of this model organism to genomic studies will begin to be revealed.

Polyploids of Brassicaceae: Genomic Insights and Assembly Strategies.

The Brassicaceae family is distinguished by its inclusion of high-value crops such as cabbage, broccoli, mustard, and wasabi, all noted for their glucosinolates. In this family, many polyploidy species are distributed and shaped by numerous whole-genome duplications, independent genome doublings, and hybridization events. The evolutionary trajectory of the family is marked by enhanced diversification and lineage splitting after paleo- and meso-polyploidization, with discernible remnants of whole-genome duplications within their genomes. The recent neopolyploidization events notably increased the proportion of polyploid species within the family. Although sequencing efforts for the Brassicaceae genome have been robust, accurately distinguishing sub-genomes remains a significant challenge, frequently complicating the assembly process. Assembly strategies include comparative analyses with ancestral species and examining k-mers, long terminal repeat retrotransposons, and pollen sequencing. This review comprehensively explores the unique genomic characteristics of the Brassicaceae family, with a particular emphasis on polyploidization events and the latest strategies for sequencing and assembly. This review will significantly improve our understanding of polyploidy in the Brassicaceae family and assist in future genome assembly methods.

Can long-read sequencing tackle the barriers, which the next-generation could not? A review.

The large-scale heterogeneity of genetic diseases necessitated the deeper examination of nucleotide sequence alterations enhancing the discovery of new targeted drug attack points. The appearance of new sequencing techniques was essential to get more interpretable genomic data. In contrast to the previous short-reads, longer lengths can provide a better insight into the potential health threatening genetic abnormalities. Long-reads offer more accurate variant identification and genome assembly methods, indicating advances in nucleotide deflect-related studies. In this review, we introduce the historical background of sequencing technologies and show their benefits and limits, as well. Furthermore, we highlight the differences between short- and long-read approaches, including their unique advances and difficulties in methodologies and evaluation. Additionally, we provide a detailed description of the corresponding bioinformatics and the current applications.

High-fidelity (repeat) consensus sequences from short reads using combined read clustering and assembly

BackgroundDespite the many cheap and fast ways to generate genomic data, good and exact genome assembly is still a problem, with especially the repeats being vastly underrepresented and often misassembled. As short reads in low coverage are already sufficient to represent the repeat landscape of any given genome, many read cluster algorithms were brought forward that provide repeat identification and classification. But how can trustworthy, reliable and representative repeat consensuses be derived from unassembled genomes?ResultsHere, we combine methods from repeat identification and genome assembly to derive these robust consensuses. We test several use cases, such as (1) consensus building from clustered short reads of non-model genomes, (2) from genome-wide amplification setups, and (3) specific repeat-centred questions, such as the linked vs. unlinked arrangement of ribosomal genes. In all our use cases, the derived consensuses are robust and representative. To evaluate overall performance, we compare our high-fidelity repeat consensuses to RepeatExplorer2-derived contigs and check, if they represent real transposable elements as found in long reads. Our results demonstrate that it is possible to generate useful, reliable and trustworthy consensuses from short reads by a combination from read cluster and genome assembly methods in an automatable way.ConclusionWe anticipate that our workflow opens the way towards more efficient and less manual repeat characterization and annotation, benefitting all genome studies, but especially those of non-model organisms.

Representing true plant genomes: haplotype-resolved hybrid pepper genome with trio-binning.

As sequencing costs decrease and availability of high fidelity long-read sequencing increases, generating experiment specific de novo genome assemblies becomes feasible. In many crop species, obtaining the genome of a hybrid or heterozygous individual is necessary for systems that do not tolerate inbreeding or for investigating important biological questions, such as hybrid vigor. However, most genome assembly methods that have been used in plants result in a merged single sequence representation that is not a true biologically accurate representation of either haplotype within a diploid individual. The resulting genome assembly is often fragmented and exhibits a mosaic of the two haplotypes, referred to as haplotype-switching. Important haplotype level information, such as causal mutations and structural variation is therefore lost causing difficulties in interpreting downstream analyses. To overcome this challenge, we have applied a method developed for animal genome assembly called trio-binning to an intra-specific hybrid of chili pepper (Capsicum annuum L. cv. HDA149 x Capsicum annuum L. cv. HDA330). We tested all currently available softwares for performing trio-binning, combined with multiple scaffolding technologies including Bionano to determine the optimal method of producing the best haplotype-resolved assembly. Ultimately, we produced highly contiguous biologically true haplotype-resolved genome assemblies for each parent, with scaffold N50s of 266.0 Mb and 281.3 Mb, with 99.6% and 99.8% positioned into chromosomes respectively. The assemblies captured 3.10 Gb and 3.12 Gb of the estimated 3.5 Gb chili pepper genome size. These assemblies represent the complete genome structure of the intraspecific hybrid, as well as the two parental genomes, and show measurable improvements over the currently available reference genomes. Our manuscript provides a valuable guide on how to apply trio-binning to other plant genomes.

The Utilization of Reference-Guided Assembly and In Silico Libraries Improves the Draft Genome of Clarias batrachus and Culter alburnus.

Long-read sequencing technologies can generate highly contiguous genome assemblies compared to short-read methods. However, their higher cost often poses a significant barrier. To address this, we explore the utilization of mapping-based genome assembly and reference-guided assembly as cost-effective alternative approaches. We assess the efficacy of these approaches in improving the contiguity of Clarias batrachus and Culter alburnus draft genomes. Our findings demonstrate that employing an iterative mapping strategy leads to a reduction in assembly errors. Specifically, after three iterations, the Mismatches per 100 kbp value for the C. batrachus genome decreased from 2447.20 to 2432.67, reaching a minimum of 2422.67 after two iterations. Additionally, the N50 value for the C. batrachus genome increased from 362,143 to 1,315,126bp, with a maximum of 1,315,403bp after two iterations. Furthermore, we achieved Mismatches per 100 kbp values of 3.70 for the reference-guided assembly of C. batrachus and 0.34 for C. alburnus. Correspondingly, the N50 value for the C. batrachus and C. alburnus genomes increased from 362,143bp and 3,686,385bp to 2,026,888bp and 43,735,735bp, respectively. Finally, we successfully utilized the improved C. batrachus and C. alburnus genomes to compare genome studies using the combined approach of Ragout and Ragtag. Through a comprehensive comparative analysis of mapping-based and reference-guided genome assembly methods, we shed light on the specific contributions of reference-guided assembly in reducing assembly errors and improving assembly continuity and integrity. These advancements establish reference-guided assembly and the utilization of in silico libraries as a promising and suitable approach for comparative genomics studies.

AgAnimalGenomes: browsers for viewing and manually annotating farm animal genomes.

Current genome sequencing technologies have made it possible to generate highly contiguous genome assemblies for non-model animal species. Despite advances in genome assembly methods, there is still room for improvement in the delineation of specific gene features in the genomes. Here we present genome visualization and annotation tools to support seven livestock species (bovine, chicken, goat, horse, pig, sheep, and water buffalo), available in a new resource called AgAnimalGenomes. In addition to supporting the manual refinement of gene models, these browsers provide visualization tracks for hundreds of RNAseq experiments, as well as data generated by the Functional Annotation of Animal Genomes (FAANG) Consortium. For species with predicted gene sets from both Ensembl and RefSeq, the browsers provide special tracks showing the thousands of protein-coding genes that disagree across the two gene sources, serving as a valuable resource to alert researchers to gene model issues that may affect data interpretation. We describe the data and search methods available in the new genome browsers and how to use the provided tools to edit and create new gene models.

The revised reference genome of the leopard gecko (Eublepharis macularius) provides insight into the considerations of genome phasing and assembly.

Genomic resources across squamate reptiles (lizards and snakes) have lagged behind other vertebrate systems and high-quality reference genomes remain scarce. Of the 23 chromosome-scale reference genomes across the order, only 12 of the ~60 squamate families are represented. Within geckos (infraorder Gekkota), a species-rich clade of lizards, chromosome-level genomes are exceptionally sparse representing only two of the seven extant families. Using the latest advances in genome sequencing and assembly methods, we generated one of the highest-quality squamate genomes to date for the leopard gecko, Eublepharis macularius (Eublepharidae). We compared this assembly to the previous, short-read only, E. macularius reference genome published in 2016 and examined potential factors within the assembly influencing contiguity of genome assemblies using PacBio HiFi data. Briefly, the read N50 of the PacBio HiFi reads generated for this study was equal to the contig N50 of the previous E. macularius reference genome at 20.4 kilobases. The HiFi reads were assembled into a total of 132 contigs, which was further scaffolded using HiC data into 75 total sequences representing all 19 chromosomes. We identified 9 of the 19 chromosomal scaffolds were assembled as a near-single contig, whereas the other 10 chromosomes were each scaffolded together from multiple contigs. We qualitatively identified that the percent repeat content within a chromosome broadly affects its assembly contiguity prior to scaffolding. This genome assembly signifies a new age for squamate genomics where high-quality reference genomes rivaling some of the best vertebrate genome assemblies can be generated for a fraction of previous cost estimates. This new E. macularius reference assembly is available on NCBI at JAOPLA010000000.

A survey of current methods to detect and genotype inversions.

Polymorphic inversions are ubiquitous in humans and they have been linked to both adaptation and disease. Following their discovery in Drosophila more than a century ago, inversions have proved to be more elusive than other structural variants. A wide variety of methods for the detection and genotyping of inversions have recently been developed: multiple techniques based on selective amplification by PCR, short- and long-read sequencing approaches, principal component analysis of small variant haplotypes, template strand sequencing, optical mapping, and various genome assembly methods. Many methods apply complex wet lab protocols or increasingly refined bioinformatic analyses. This review is an attempt to provide a practical summary and comparison of the methods that are in current use, with a focus on metrics such as the maximum size of segmental duplications at inversion breakpoints that each method can tolerate, the size range of inversions that they recover, their throughput, and whether the locations of putative inversions must be known beforehand.

Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing

Obtaining complete phytoplasma genomes is difficult due to the lack of a culture system for these bacteria. To improve genome assembly, a non-ionic, low- and iso-osmotic iodixanol (Optiprep™) density gradient centrifugation method was developed to enrich for phytoplasma cells and deplete plant host tissues prior to deoxyribonucleic acid (DNA) extraction and high-throughput sequencing (HTS). After density gradient enrichment, potato infected with a ‘Candidatus Phytoplasma australasia’-related strain showed a ∼14-fold increase in phytoplasma HTS reads, with a ∼1.7-fold decrease in host genomic reads compared to the DNA extracted from the same sample without density gradient centrifugation enrichment. Additionally, phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment. The method was repeated on capsicum infected with Sweet Potato Little Leaf phytoplasma (‘Ca. Phytoplasma australasia’-related strain) with a lower phytoplasma titer than the potato. In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained, with the genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment. Phytoplasmas from potato and capsicum were both enriched at a density of 1.049–1.058 g/ml. Finally, we present two highly contiguous ‘Ca. Phytoplasma australasia’ phytoplasma reference genomes sequenced from naturally infected Solanaceae hosts in Australia. Obtaining high-quality phytoplasma genomes from naturally infected hosts will improve insights into phytoplasma taxonomy, which will improve their detection and disease management.

The first genome for the Cape Primrose Streptocarpus rexii (Gesneriaceae), a model plant for studying meristem-driven shoot diversity.

Cape Primroses (Streptocarpus, Gesneriaceae) are an ideal study system for investigating the genetics underlying species diversity in angiosperms. Streptocarpus rexii has served as a model species for plant developmental research for over five decades due to its unusual extended meristem activity present in the leaves. In this study, we sequenced and assembled the complete nuclear, chloroplast, and mitochondrial genomes of S. rexii using Oxford Nanopore Technologies long read sequencing. Two flow cells of PromethION sequencing resulted in 32 billion reads and were sufficient to generate a draft assembly including the chloroplast, mitochondrial and nuclear genomes, spanning 776 Mbp. The final nuclear genome assembly contained 5,855 contigs, spanning 766 Mbp of the 929‐Mbp haploid genome with an N50 of 3.7 Mbp and an L50 of 57 contigs. Over 70% of the draft genome was identified as repeats. A genome repeat library of Gesneriaceae was generated and used for genome annotation, with a total of 45,045 genes annotated in the S. rexii genome. Ks plots of the paranomes suggested a recent whole genome duplication event, shared between S. rexii and Primulina huaijiensis. A new chloroplast and mitochondrial genome assembly method, based on contig coverage and identification, was developed, and successfully used to assemble both organellar genomes of S. rexii . This method was developed into a pipeline and proved widely applicable. The nuclear genome of S. rexii and other datasets generated and reported here will be invaluable resources for further research to aid in the identification of genes involved in morphological variation underpinning plant diversification.

Chasing perfection: validation and polishing strategies for telomere-to-telomere genome assemblies.

Advances in long-read sequencing technologies and genome assembly methods have enabled the recent completion of the first telomere-to-telomere human genome assembly, which resolves complex segmental duplications and large tandem repeats, including centromeric satellite arrays in a complete hydatidiform mole (CHM13). Although derived from highly accurate sequences, evaluation revealed evidence of small errors and structural misassemblies in the initial draft assembly. To correct these errors, we designed a new repeat-aware polishing strategy that made accurate assembly corrections in large repeats without overcorrection, ultimately fixing 51% of the existing errors and improving the assembly quality value from 70.2 to 73.9 measured from PacBio high-fidelity and Illumina k-mers. By comparing our results to standard automated polishing tools, we outline common polishing errors and offer practical suggestions for genome projects with limited resources. We also show how sequencing biases in both high-fidelity and Oxford Nanopore Technologies reads cause signature assembly errors that can be corrected with a diverse panel of sequencing technologies.

Full-Likelihood Genomic Analysis Clarifies a Complex History of Species Divergence and Introgression: The Example of the erato-sara Group of Heliconius Butterflies.

Introgressive hybridization plays a key role in adaptive evolution and species diversification in many groups of species. However, frequent hybridization and gene flow between species make estimation of the species phylogeny and key population parameters challenging. Here, we show that by accounting for phasing and using full-likelihood methods, introgression histories and population parameters can be estimated reliably from whole-genome sequence data. We employ the multispecies coalescent (MSC) model with and without gene flow to infer the species phylogeny and cross-species introgression events using genomic data from six members of the erato-sara clade of Heliconius butterflies. The methods naturally accommodate random fluctuations in genealogical history across the genome due to deep coalescence. To avoid heterozygote phasing errors in haploid sequences commonly produced by genome assembly methods, we process and compile unphased diploid sequence alignments and use analytical methods to average over uncertainties in heterozygote phase resolution. There is robust evidence for introgression across the genome, both among distantly related species deep in the phylogeny and between sister species in shallow parts of the tree. We obtain chromosome-specific estimates of key population parameters such as introgression directions, times and probabilities, as well as species divergence times and population sizes for modern and ancestral species. We confirm ancestral gene flow between the sara clade and an ancestral population of Heliconius telesiphe, a likely hybrid speciation origin for Heliconius hecalesia, and gene flow between the sister species Heliconius erato and Heliconius himera. Inferred introgression among ancestral species also explains the history of two chromosomal inversions deep in the phylogeny of the group. This study illustrates how a full-likelihood approach based on the MSC makes it possible to extract rich historical information of species divergence and gene flow from genomic data. [3s; bpp; gene flow; Heliconius; hybrid speciation; introgression; inversion; multispecies coalescent]

Signatures of selection and evolutionary relevance of cytochrome P450s in plant-insect interactions.

Enzymes in the cytochrome P450 (P450) superfamily have important functions ranging from those that are essential for the physiology and development of the individual to those that mediate interactions between individuals and their biotic environment. Until recently the study of P450s had focused on single functions, substrates, or pathways. Recent advances in sequencing, genome assembly, and phylogenetic methods have returned emphasis to the adaptive value of these enzymes in the context of herbivory. Comparisons of whole repertoires of P450s across related species reveal that P450s capable of metabolizing xenobiotics have an increased rate of gains compared to losses after gene duplications. In plants, studies have focused on enzymes and end-functions that have converged to provide increased resistance to herbivory. This review summarizes the latest findings related to the ecological value of P450s in the interactions between phytophagous insects and their host plants.

Assembly of chromosome-scale contigs by efficiently resolving repetitive sequences with long reads

The abundant repetitive sequences in complex eukaryotic genomes cause fragmented assemblies, which lose value as reference genomes, often due to incomplete gene sequences and unanchored or mispositioned contigs on chromosomes. Here we report a genome assembly method HERA, which resolves repeats efficiently by constructing a connection graph from an overlap graph. We test HERA on the genomes of rice, maize, human, and Tartary buckwheat with single-molecule sequencing and mapping data. HERA correctly assembles most of the previously unassembled regions, resulting in dramatically improved, highly contiguous genome assemblies with newly assembled gene sequences. For example, the maize contig N50 size reaches 61.2 Mb and the Tartary buckwheat genome comprises only 20 contigs. HERA can also be used to fill gaps and fix errors in reference genomes. The application of HERA will greatly improve the quality of new or existing assemblies of complex genomes.

First report of Burkholderia pseudomallei ST412 and ST734 clones harbouring blaOXA-57 but susceptible to imipenem in India.

Melioidosis caused by Burkholderia pseudomallei has become an important clinical threat, especially in Northern Australia and Southeast Asia. However, the genome information on this pathogen is limited. B. pseudomallei isolates identified from bloodstream infections from inpatients were subjected to whole-genome sequencing by IonTorrent PGM and MinION Oxford Nanopore sequencing technologies. Highly accurate complete genomes of two strains, VB3253 and VB2514, were obtained by a hybrid genome assembly method using both short and long DNA reads. Both isolates carried blaPenI and carbapenemase-encoding blaOXA-57 genes, although the isolates were susceptible to imipenem by E-test method with MIC 1 μg/mL. Multiple IS family transposases specific for all non-fermenting Gram-negative bacteria (NFGNBs)—especially IS3 and IS5, which facilitate mobilization of extended-spectrum β-lactamase (ESBL) and carbapenemase genes—were carried in these genomes. This further adds to the complexity of gene transmission. These IS families were identified only upon hybrid genome assembly and would otherwise be missed.

A comparative analysis of methods for de novo assembly of hymenopteran genomes using either haploid or diploid samples

Diverse invertebrate taxa including all 200,000 species of Hymenoptera (ants, bees, wasps and sawflies) have a haplodiploid sex determination system, where females are diploid and males are haploid. Thus, hymenopteran genome projects can make use of DNA from a single haploid male sample, which is assumed advantageous for genome assembly. For the purpose of gene annotation, transcriptome sequencing is usually conducted using RNA from a pool of individuals. We conducted a comparative analysis of genome and transcriptome assembly and annotation methods, using genetic sources of different ploidy: (1) DNA from a haploid male or a diploid female (2) RNA from the same haploid male or a pool of individuals. We predicted that the use of a haploid male as opposed to a diploid female will simplify the genome assembly and gene annotation thanks to the lack of heterozygosity. Using DNA and RNA from the same haploid individual is expected to provide better confidence in transcript-to-genome alignment, and improve the annotation of gene structure in terms of the exon/intron boundaries. The haploid genome assemblies proved to be more contiguous, with both contig and scaffold N50 size at least threefold greater than their diploid counterparts. Completeness evaluation showed mixed results. The SOAPdenovo2 diploid assembly was missing more genes than the haploid assembly. The SPAdes diploid assembly had more complete genes, but a higher level of duplicates, and a greatly overestimated genome size. When aligning the two transcriptomes against the male genome, the male transcriptome gave 2–3% more complete transcripts than the pool transcriptome for genes with comparable expression levels in both transcriptomes. However, this advantage disappears in the final results of the gene annotation pipeline that incorporates evidence from homologous proteins. The RNA pool is still required to obtain the full transcriptome with genes that are expressed in other life stages and castes. In conclusion, the use of a haploid source material for a de novo genome project provides a substantial advantage to the quality of the genome draft and the use of RNA from the same haploid individual for transcriptome to genome alignment provides a minor advantage for genes that are expressed in the adult male.

Progress in plant genome sequencing: research directions

Since the first plant genome of Arabidopsis thaliana has been sequenced and published, genome sequencing technologies have undergone significant changes. New algorithms, sequencing technologies and bioinformatic approaches were adopted to obtain genome, transcriptome and exome sequences for model and crop species, which have permitted deep inferences into plant biology. As a result of an improved genome assembly and analysis methods, genome sequencing costs plummeted and the number of high-quality plant genome sequences is constantly growing. Consequently, more than 300 plant genome sequences have been published over the past twenty years. Although many of the published genomes are considered incomplete, they proved to be a valuable tool for identifying genes involved in the formation of economically valuable plant traits, for marker-assisted and genomic selection and for comparative analysis of plant genomes in order to determine the basic patterns of origin of various plant species. Since a high coverage and resolution of a genome sequence is not enough to detect all changes in complex samples, targeted sequencing, which consists in the isolation and sequencing of a specific region of the genome, has begun to develop. Targeted sequencing has a higher detection power (the ability to identify new differences/variants) and resolution (up to one basis). In addition, exome sequencing (the method of sequencing only protein-coding genes regions) is actively developed, which allows for the sequencing of non-expressed alleles and genes that cannot be found with RNA-seq. In this review, an analysis of sequencing technologies development and the construction of “reference” genomes of plants is performed. A comparison of the methods of targeted sequencing based on the use of the reference DNA sequence is accomplished.

Comparative analysis of de novo assembly of Bacillus cereus HUB4-4 short reads

The automated Sanger method is considered as a first generation technology and newer methods are referred to as next generation sequencing. These newer technologies constitute various strategies that rely on a combination of template preparation sequencing and imaging and genome alignment and assembly methods. NGS or high throughput sequencing technologies are intended to lower the cost of DNA sequencing beyond what is possible with standard dye terminator methods. Bacillus cereus is a very common dirt-dwelling bacterium which can survive in an oxygen environment with the help of protective endospores. There are several strains like HUB4-4,HUB2-3, CER057etc are cause harm to humans, whereas others are used as helpful probiotics. De novo assembly is mainly used for the purpose of assembling short reads to create full-length sequences. Bacillus cereus HUB4-4 for this organism there is no contigs and scaffolds are available. So De novo assembly was carried out to find scaffolds and contigs by using the CLC Genomics Workbench and DRA pipeline and N25, N50, N75 contigs were determined. For identified N50 contigs, BLAST is done at NCBI and identified related organism toBacillus cereus HUB4-4 in CLC genomics. N50 contigs in DRA pipeline were also determined by using three software tools like soap de novo, velvet, abyss for the Bacillus cereus HUB4-4 data using short read assembly for paired end data and compare the output of these two tools and identified CLC genomics workbench is more efficient.