Hierarchical Genome Assembly Process Research Articles

Whole-Genome Sequence and Pathogenicity Analysis of Providencia Heimbachae Causing Diarrhea in Weaned Piglets.

Providencia heimbachae was previously identified in piglets with post-weaned diarrhea and associated with hindlimb paralysis. However, the pathogenic mechanisms and virulence factors of P. heimbachae are not fully known. Whole-genome sequence analysis will be helpful to extend our understanding of the characterization of P. heimbachae at a genomic level. In this study, we sequenced the whole genome of P. heimbachae for the first time using PacBio RS II sequencers and assembled de novo through hierarchical genome assembly process (HGAP). Furthermore, we performed further genome annotation. The genome of P. heimbachae 99101 consists of a circular chromosome (4,262,828bp) and a circular plasmid (231,957bp) with G + C contents of 40.43 and 47.16%, respectively. Genome-wide sequence analysis yielded a total of 286 predicted virulence factors, 178 resistance genes, 17 chaperone protein manipulators of fimbriae, 47 genes involved in the encoding of flagellin, 12 cell membrane-associated virulence genes, 18 Enterobacteriaceae common antigens, etc. Based on genome analysis, we preliminarily confirmed through animal experiments that the capsule was the virulence factor of P. heimbachae causing hindlimb paralysis in animals. Our study provides a genetic basis for further elucidation of the characteristics and functional mechanisms of P. heimbachae as a conditionally pathogenic bacterium, as well as a direction for research into the mechanism of action of P. heimbachae infecting humans, extending knowledge of P. heimbachae as an important zoonotic pathogen.

Draft genome sequence and probiotic functional property analysis of Lactobacillus gasseri LM1065 for food industry applications

Probiotics are defined as live organisms in the host that contribute to health benefits. Lactobacillus gasseri LM1065, isolated from human breast milk, was investigated for its probiotic properties based on its genome. Draft genome map and de novo assembly were performed using the PacBio RS II system and hierarchical genome assembly process (HGAP). Probiotic properties were determined by the resistance to gastric conditions, adherence ability, enzyme production, safety assessment and mobile genetic elements. The fungistatic effect and inhibition of hyphae transition were studied using the cell-free supernatant (CFS). L. gasseri LM1065 showed high gastric pepsin tolerance and mild tolerance to bile salts. Auto-aggregation and hydrophobicity were measured to be 61.21% and 61.55%, respectively. The adherence to the human intestinal epithelial cells was measured to be 2.02%. Antibiotic-resistance genes and putative virulence genes were not predicted in the genomic analysis, and antibiotic susceptibility was satisfied by the criteria of the European Food Safety Authority. CFS showed a fungistatic effect and suppressed the tricarboxylic acid cycle in Candida albicans (29.02%). CFS also inhibited the transition to true hyphae and damaged the blastoconidia. This study demonstrates the essential properties of this novel probiotic, L. gasseri LM1065, and potential to inhibit vaginal C. albicans infection.

Complete Genome Sequence of Vibrio maritimus BH16, a Siderophore-Producing Mutualistic Bacterium Isolated from Diatom Skeletonema costatum.

Complete Genome Sequence of Vibrio maritimus BH16, a Siderophore-Producing Mutualistic Bacterium Isolated from Diatom Skeletonema costatum.

Antibiotic Biosynthesis Pathways from Endophytic Streptomyces SUK 48 through Metabolomics and Genomics Approaches.

Streptomyces sp. has been known to be a major antibiotic producer since the 1940s. As the number of cases related to resistance pathogens infection increases yearly, discovering the biosynthesis pathways of antibiotic has become important. In this study, we present the streamline of a project report summary; the genome data and metabolome data of newly isolated Streptomyces SUK 48 strain are also analyzed. The antibacterial activity of its crude extract is also determined. To obtain genome data, the genomic DNA of SUK 48 was extracted using a commercial kit (Promega) and sent for sequencing (Pac Biosciences technology platform, Menlo Park, CA, USA). The raw data were assembled and polished using Hierarchical Genome Assembly Process 4.0 (HGAP 4.0). The assembled data were structurally predicted using tRNAscan-SE and rnammer. Then, the data were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) database and antiSMASH analysis. Meanwhile, the metabolite profile of SUK 48 was determined using liquid chromatography-mass spectrophotometry (LC-MS) for both negative and positive modes. The results showed that the presence of kanamycin and gentamicin, as well as the other 11 antibiotics. Nevertheless, the biosynthesis pathways of aurantioclavine were also found. The cytotoxicity activity showed IC50 value was at 0.35 ± 1.35 mg/mL on the cell viability of HEK 293. In conclusion, Streptomyces sp. SUK 48 has proven to be a non-toxic antibiotic producer such as auranticlavine and gentamicin.

Genome based phylogeny and virulence factor analysis of mastitis causing Escherichia coli isolated from Indian cattle

Mastitis is a highly infectious disease prevalent in dairy cattle and it is majorly caused by Escherichia coli (E. coli). The objective of present study is to investigate the occurrence of virulence genes, antimicrobial susceptibility and comparative analysis of E. coli (IVRI KOL CP4 and CM IVRI KOL-1) isolates from mastitis infected animal. Whole-genome sequencing (WGS) was performed using a PacBio RS II system and de novo assembled using Hierarchical Genome Assembly Process (HGAP3). Bacterial Pan Genome Analysis Pipeline (BPGA) was used for pangenome analysis. A set of 50 E. coli isolates were used for comparative analysis (48 collected from the database and 2 reference sequences). Core genes were further concatenated for phylogenetic analyses. In silico analysis was performed for antibiotic resistance and virulence gene identification. Both of the E. coli isolates carried many resistance genes including, b-lactamase, quinolones, rifampicin, macrolide, aminoglycoside and phenicols resistance. We detected 39 virulence genes in IVRI KOL CP4 and 52 in CM IVRI KOL-1 which include toxins, adhesions, invasins, secretion machineries or iron acquisition system. High prevalence of mastitis strains belongs to phylogroups A, although few isolates were also assigned to phylogenetic groups B1 and B2. In conclusion, the present study reported the presence of genes involved in Adherence, Iron acquisition, secretion system and toxins which shown to be crucial in MPEC pathogenicity. This is the first whole genome analysis of MPEC strains to be carried out in Indian isolate to highlights the spread of resistance and virulence genes in food animals.

Genomic insights into a complete deletion of the mgrB locus in colistin-resistant Klebsiella pneumoniae ST2268 isolated from a human infection

The emergence of colistin-resistant Klebsiella pneumoniae (CoRKp) is a serious public-health issue as colistin is the last line of defence against infections caused by multidrug-resistant Gram-negative bacteria. In this study, we generated a draft genome sequence for CoRKp strain P094-1 isolated from a sputum sample of an infected patient. Whole genomic DNA of strain P094-1 was sequenced using a PacBio sequencing platform. Generated reads were de novo assembled using Hierarchical Genome Assembly Process (HGAP) v.3.0. Colistin resistance-related genes were predicted from the genome sequence and were validated experimentally. The genome of strain P094-1 lacked a 20.3-kb region, including complete deletion of the mgrB gene. Molecular and genome sequencing-based analyses revealed that the observed colistin resistance of P094-1 could not be attributed to plasmid-borne genes mcr-1 to mcr-9 or to alteration of the pmr and pho operons (deletions, insertions or substitutions), but was conferred by an insertion sequence 1 (IS1)-induced total deletion of mgrB. This is the first reported whole-genome sequence of an unusual CoRKp isolate containing an IS1-induced deletion of mgrB.

Genome-scale analyses and characteristics of putative pathogenicity genes of Stagonosporopsis cucurbitacearum, a pumpkin gummy stem blight fungus

Outbreaks of gummy stem blight (GSB), an emerging seed pumpkin disease, have increased in number and have become more widespread in recent years. Previously we reported that Stagonosporopsis cucurbitacearum (Sc.) is the dominant fungal cause of pumpkin seedling GSB in Northeast China, where it has greatly reduced crop yields in that region. Here, high-throughput whole-genome sequencing and assembly of the Sc. genome were conducted toward revealing pathogenic molecular regulatory mechanisms involved in fungal growth and development. Zq-1 as representative Sc. strain, DNA of Zq-1was prepared for genomic sequencing, we obtained 5.24 Gb of high-quality genomic sequence data via PacBio RS II sequencing. After sequence data was processed to filter out low quality reads, a hierarchical genome-assembly process was employed that generated a genome sequence of 35.28 Mb in size. A total of 9844 genes were predicted, including 237 non-coding RNAs, 1024 genes encoding proteins with signal peptides, 2066 transmembrane proteins and 756 secretory proteins.Transcriptional identification revealed 54 differentially expressed secretory proteins. Concurrently, 605, 130 and 2869 proteins were matched in the proprietary databases Carbohydrate-Active EnZymes database (CAZyme), Transporter Classification Database (TCDB) and Pathogen–Host Interactions database (PHI), respectively. And 96 and 36 DEGs were identified form PHI database and CAZyme database, respectively. In addition, contig00011.93 was an up-regulated DEG involving ATP-binding cassette metabolism in the procession of infection. In order to test relevance of gene predictions to GSB, DEGs with potential pathogenic relevance were revealed through transcriptome data analysis of Sc. strains pre- and post-infection of pumpkin. Interestingly, Sc. and Leptosphaeria maculans (Lm.) exhibited relatively similar with genome lengths, numbers of protein-coding genes and other characteristics. This work provides a foundation for future exploration of additional Sc. gene functions toward the development of more effective GSB control strategies.

Complete genome sequence of the halophilic PHA-producing bacterium Halomonas sp. SF2003: insights into its biotechnological potential.

A halophilic Gram-negative eubacterium was isolated from the Iroise Sea and identified as an efficient producer of polyhydroxyalkanoates (PHA). The strain, designated SF2003, was found to belong to the Halomonas genus on the basis of 16S rRNA gene sequence similarity. Previous biochemical tests indicated that the Halomonas sp. strain SF2003 is capable of supporting various culture conditions which sometimes can be constraining for marine strains. This versatility could be of great interest for biotechnological applications. Therefore, a complete bacterial genome sequencing and de novo assembly were performed using a PacBio RSII sequencer and Hierarchical Genome Assembly Process software in order to predict Halomonas sp. SF2003 metabolisms, and to identify genes involved in PHA production and stress tolerance. This study demonstrates the complete genome sequence of Halomonas sp. SF2003 which contains a circular 4,36 Mbp chromosome, and replaces the strain in a phylogenetic tree. Genes related to PHA metabolism, carbohydrate metabolism, fatty acid metabolism and stress tolerance were identified and a comparison was made with metabolisms of relative species. Genes annotation highlighted the presence of typical genes involved in PHA biosynthesis such as phaA, phaB and phaC and enabled a preliminary analysis of their organization and characteristics. Several genes of carbohydrates and fatty acid metabolisms were also identified which provided helpful insights into both a better knowledge of the intricacies of PHA biosynthetic pathways and of production purposes. Results show the strong versatility of Halomonas sp. SF2003 to adapt to various temperatures and salinity which can subsequently be exploited for industrial applications such as PHA production.

Whole genome sequence of Bacillus thuringiensis ATCC 10792 and improved discrimination of Bacillus thuringiensis from Bacillus cereus group based on novel biomarkers

BackgroundAmong the Bacillus cereus group, B. thuringiensis, is one of the most extensively used biological control agent. The present study reports the complete genome and four novel plasmid analysis of the type strain B. thuringiensis ATCC 10792. MethodsComplete genome sequencing of Bacillus thuringiensis ATCC 10792, assembled using de-novo (v.3.2.0, assembly name MIRA3), Pac-Bio sequencers and Hierarchical Genome Assembly Process software (version 4.1) and real-time polymerase chain reaction (qPCR) is a consistent technique for quantifying gene expression based on specific biomarkers, in addition the efficiency of the primers were analysed based on artificially spiked food samples on lettuce, kimbab and spinach with B. thuringiensis ATCC 10792. ResultsComplete genome annotation was performed, and a total of 6269 proteins with 5427594 bps were identified and four novel plasmid (poh2, poh3, poh4, poh5) a total of 134, 131, 96, 21 proteins with 113294; 92,949; 86488; 11332 bps were identified. Six selective genes (lipoprotein-lipo, methyltransferase-MT, S-layer homology domain protein-BC, flagellar motor protein-motB, transcriptional regulator-XRE, crystal protein-cry2) and associated four novel plasmids were investigated along with the characteristics and expression profiles of two housekeeping genes (chaperonin protein-GroEL and topoisomerase enzyme-gyrB). Although from the assessment of 120 strains, both GroEL and gyrB showed 100% specificity towards detection of both B. thuringiensis in artificially spiked vegetable samples. All the eight genes revealed no specificity towards any of the 9 non- Bacillus strains. ConclusionIn our study based on the complete genome and plasmid sequence of B. thuringiensis ATCC 10792, among the six discriminating genes, specifically GroEL, gyrB and XRE showed promising results in identifying B. thuringiensis ATCC 10792, and there detection limit was 3.0–9.6 log CFU/g in the food samples respectfully. The key role in control of the predatory biological agent.

In silico genome analysis reveals the metabolic versatility and biotechnology potential of a halotorelant phthalic acid esters degrading Gordonia alkanivorans strain YC-RL2

Members of genus Gordonia are known to degrade various xenobitics and produce secondary metabolites. The genome of a halotorelant phthalic acid ester (PAEs) degrading actinobacterium Gordonia alkanivorans strain YC-RL2 was sequenced using Biosciences RS II platform and Single Molecular Real-Time (SMRT) technology. The reads were assembled de novo by hierarchical genome assembly process (HGAP) algorithm version 2. Genes were annotated by NCBI Prokaryotic Genome Annotation Pipeline. The generated genome sequence was 4,979,656 bp with an average G+C content of 67.45%. Calculation of ANI confirmed previous classification that strain YC-RL2 is G. alkanivorans. The sequences were searched against KEGG and COG databases; 3132 CDSs were assigned to COG families and 1808 CDSs were predicted to be involved in 111 pathways. 95 of the KEGG annotated genes were predicted to be involved in the degradation of xenobiotics. A phthalate degradation operon could not be identified in the genome indicating that strain YC-RL2 possesses a novel way of phthalate degradation. A total of 203 and 22 CDSs were annotated as esterase/hydrolase and dioxygenase genes respectively. A total of 53 biosynthetic gene clusters (BGCs) were predicted by antiSMASH (antibiotics & Secondary Metabolite Analysis Shell) bacterial version 4.0. The genome also contained putative genes for heavy metal metabolism. The strain could tolerate 1 mM of Cd2+, Co2+, Cu2+, Ni2+, Zn2+, Mn2+ and Pb2+ ions. These results show that strain YC-RL2 has a great potential to degrade various xenobiotics in different environments and will provide a rich genetic resource for further biotechnological and remediation studies.

New perspectives on Mega plasmid sequence (poh1) in Bacillus thuringiensis ATCC 10792 harbouring antimicrobial, insecticidal and antibiotic resistance genes

Bacillus thuringiensis promotes the growth of numerous economically important crops. The present study presents the complete genome sequence for a mega plasmid present in the type strain of B. thuringiensis ATCC 10792, a typical spore-forming Gram-positive bacterium with insecticidal activity, and investigates its genetic characteristics. The genome was sequenced and assembled de novo using Pac-Bio sequencers and the Hierarchical Genome Assembly Process, respectively. Further genome annotation was performed, and a total of 489 proteins and a novel mega-plasmid (poh1) with 584,623 bps were identified. The organization of poh1 revealed the genes involved in the insecticidal toxin pathway. The genes responsible for antimicrobial, insecticidal and antibiotic activities were well conserved in poh1, indicating an intimate association with plant hosts. The poh1 plasmid contains the gene encoding a novel crystal protein kinase responsible for production of zeta toxin, which poisons insects and other Gram-negative bacteria through the global inhibition of peptidoglycan synthesis. Lantibiotics are a group of bacteriocins that include the biologically active antimicrobial peptide Paenibacillin. Further, poh1 also contains the genes that encode the gramicidin S prototypical antibiotic peptide and tetracycline resistance protein. In conclusion, the strain-specific genes of B. thuringiensis strain ATCC 10792 were identified through complete genome sequencing and bioinformatics data based on major pathogenic factors that contribute to further studies of the pathogenic mechanism and phenotype analyses.

The impact of Staphylococcus aureus genomic variation on clinical phenotype of children with acute hematogenous osteomyelitis

BackgroundChildren with acute hematogenous osteomyelitis (AHO) have a broad spectrum of illness ranging from mild to severe. The purpose of this study is to evaluate the impact of genomic variation of Staphylococcus aureus on clinical phenotype of affected children and determine which virulence genes correlate with severity of illness. MethodsDe novo whole genome sequencing was conducted for a strain of Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA), using PacBio Hierarchical Genome Assembly Process (HGAP) from 6 Single Molecule Real Time (SMRT) Cells, as a reference for DNA library assembly of 71 Staphylococcus aureus isolates from children with AHO. Virulence gene annotation was based on exhaustive literature review and genomic data in NCBI for Staphylococcus aureus. Clinical phenotype was assessed using a validated severity score. Kruskal-Wallis rank sum test determined association between clinical severity and virulence gene presence using False Discovery Rate (FDR), significance <0.01. ResultsPacBio produced an assembled genome of 2,898,306 bp and 2054 Open Reading Frames (ORFs). Annotation confirmed 201 virulence genes. Statistical analysis of gene presence by clinical severity found 40 genes significantly associated with severity of illness (FDR ≤0.009). MRSA isolates encoded a significantly greater number of virulence genes than did MSSA (p < 0.0001). Phylogenetic analysis by maximum likelihood (PAML) demonstrated the relatedness of genomic distance to clinical phenotype. ConclusionsThe Staphylococcus aureus genome contains virulence genes which are significantly associated with severity of illness in children with osteomyelitis. This study introduces a novel reference strain and detailed annotation of Staphylococcus aureus virulence genes. While this study does not address bacterial gene expression, a platform is created for future transcriptome investigations to elucidate the complex mechanisms involved in childhood osteomyelitis.

Identification of a leucine-rich repeat receptor-like serine/threonine-protein kinase as a candidate gene for Rvi12 (Vb)-based apple scab resistance

Apple scab caused by Venturia inaequalis is the most important fungal disease of apples (Malus × domestica). Currently, the disease is controlled by up to 15 fungicide applications to the crop per year. Resistant apple cultivars will help promote the sustainable control of scab in commercial orchards. The breakdown of the Rvi6 (Vf) major-gene based resistance, the most used resistance gene in apple breeding, prompted the identification and characterization of new scab resistance genes. By using a large segregating population, the Rvi12 scab resistance gene was previously mapped to a genetic location flanked by molecular markers SNP_23.599 and SNP_24.482. Starting from these markers, utilizing chromosome walking of a Hansen’s baccata #2 (HB2) BAC-library; a single BAC clone spanning the Rvi12 interval was identified. Following Pacific Biosciences (PacBio) RS II sequencing and the use of the hierarchical genome assembly process (HGAP) assembly of the BAC clone sequence, the Rvi12 resistance locus was localized to a 62.3-kb genomic region. Gene prediction and in silico characterization identified a single candidate resistance gene. The gene, named here as Rvi12_Cd5, belongs to the LRR receptor-like serine/threonine-protein kinase family. In silico comparison of the resistance allele from HB2 and the susceptible allele from Golden Delicious (GD) identified the presence of an additional intron in the HB2 allele. Conserved domain analysis identified the presence of four additional LRR motifs in the susceptible allele compared to the resistance allele. The constitutive expression of Rvi12_Cd5 in HB2, together with its structural similarity to known resistance genes, makes it the most likely candidate for Rvi12 scab resistance in apple.

Whole Genome Sequence and Comparative Genomics of the Novel Lyme Borreliosis Causing Pathogen, Borrelia mayonii.

Borrelia mayonii, a Borrelia burgdorferi sensu lato (Bbsl) genospecies, was recently identified as a cause of Lyme borreliosis (LB) among patients from the upper midwestern United States. By microscopy and PCR, spirochete/genome loads in infected patients were estimated at 105 to 106 per milliliter of blood. Here, we present the full chromosome and plasmid sequences of two B. mayonii isolates, MN14-1420 and MN14-1539, cultured from blood of two of these patients. Whole genome sequencing and assembly was conducted using PacBio long read sequencing (Pacific Biosciences RSII instrument) followed by hierarchical genome-assembly process (HGAP). The B. mayonii genome is ~1.31 Mbp in size (26.9% average GC content) and is comprised of a linear chromosome, 8 linear and 7 circular plasmids. Consistent with its taxonomic designation as a new Bbsl genospecies, the B. mayonii linear chromosome shares only 93.83% average nucleotide identity with other genospecies. Both B. mayonii genomes contain plasmids similar to B. burgdorferi sensu stricto lp54, lp36, lp28-3, lp28-4, lp25, lp17, lp5, 5 cp32s, cp26, and cp9. The vls locus present on lp28-10 of B. mayonii MN14-1420 is remarkably long, being comprised of 24 silent vls cassettes. Genetic differences between the two B. mayonii genomes are limited and include 15 single nucleotide variations as well as 7 fewer silent vls cassettes and a lack of the lp5 plasmid in MN14-1539. Notably, 68 homologs to proteins present in B. burgdorferi sensu stricto appear to be lacking from the B. mayonii genomes. These include the complement inhibitor, CspZ (BB_H06), the fibronectin binding protein, BB_K32, as well as multiple lipoproteins and proteins of unknown function. This study shows the utility of long read sequencing for full genome assembly of Bbsl genomes, identifies putative genome regions of B. mayonii that may be linked to clinical manifestation or tissue tropism, and provides a valuable resource for pathogenicity, diagnostic and vaccine studies.

High-Quality Genome Assembly and Annotation for Plasmodium coatneyi, Generated Using Single-Molecule Real-Time PacBio Technology.

Plasmodium coatneyi is a protozoan parasite species that causes simian malaria and is an excellent model for studying disease caused by the human malaria parasite, P. falciparum. Here we report the complete (nontelomeric) genome sequence of P. coatneyi Hackeri generated by the application of only Pacific Biosciences RS II (PacBio RS II) single-molecule real-time (SMRT) high-resolution sequence technology and assembly using the Hierarchical Genome Assembly Process (HGAP). This is the first Plasmodium genome sequence reported to use only PacBio technology. This approach has proven to be superior to short-read only approaches for this species.

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome through long-read (>11 kb), single molecule, real-time sequencing.

The application of next-generation sequencing to estimate genetic diversity of Plasmodium falciparum, the most lethal malaria parasite, has proved challenging due to the skewed AT-richness [∼80.6% (A + T)] of its genome and the lack of technology to assemble highly polymorphic subtelomeric regions that contain clonally variant, multigene virulence families (Ex: var and rifin). To address this, we performed amplification-free, single molecule, real-time sequencing of P. falciparum genomic DNA and generated reads of average length 12 kb, with 50% of the reads between 15.5 and 50 kb in length. Next, using the Hierarchical Genome Assembly Process, we assembled the P. falciparum genome de novo and successfully compiled all 14 nuclear chromosomes telomere-to-telomere. We also accurately resolved centromeres [∼90–99% (A + T)] and subtelomeric regions and identified large insertions and duplications that add extra var and rifin genes to the genome, along with smaller structural variants such as homopolymer tract expansions. Overall, we show that amplification-free, long-read sequencing combined with de novo assembly overcomes major challenges inherent to studying the P. falciparum genome. Indeed, this technology may not only identify the polymorphic and repetitive subtelomeric sequences of parasite populations from endemic areas but may also evaluate structural variation linked to virulence, drug resistance and disease transmission.

Filling in the Gap of Human Chromosome 4: Single Molecule Real Time Sequencing of Macrosatellite Repeats in the Facioscapulohumeral Muscular Dystrophy Locus.

A majority of facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of macrosatellite repeats called D4Z4 that are located in the subtelomeric region of human chromosome 4q35. Sequencing the FSHD locus has been technically challenging due to its long size and nearly identical nature of repeat elements. Here we report sequencing and partial assembly of a BAC clone carrying an entire FSHD locus by a single molecule real time (SMRT) sequencing technology which could produce long reads up to about 18 kb containing D4Z4 repeats. De novo assembly by Hierarchical Genome Assembly Process 1 (HGAP.1) yielded a contig of 41 kb containing all but a part of the most distal D4Z4 element. The validity of the sequence model was confirmed by an independent approach employing anchored multiple sequence alignment by Kalign using reads containing unique flanking sequences. Our data will provide a basis for further optimization of sequencing and assembly conditions of D4Z4.

Toward Complete Bacterial Genome Sequencing Through the Combined Use of Multiple Next-Generation Sequencing Platforms.

PacBio's long-read sequencing technologies can be successfully used for a complete bacterial genome assembly using recently developed non-hybrid assemblers in the absence of secondgeneration, high-quality short reads. However, standardized procedures that take into account multiple pre-existing second-generation sequencing platforms are scarce. In addition to Illumina HiSeq and Ion Torrent PGM-based genome sequencing results derived from previous studies, we generated further sequencing data, including from the PacBio RS II platform, and applied various bioinformatics tools to obtain complete genome assemblies for five bacterial strains. Our approach revealed that the hierarchical genome assembly process (HGAP) non-hybrid assembler resulted in nearly complete assemblies at a moderate coverage of ~75x, but that different versions produced non-compatible results requiring post processing. The other two platforms further improved the PacBio assembly through scaffolding and a final error correction.

OR01: AUTOMATED ASSEMBLY OF COMPLEX IMMUNOGENETIC HAPLOTYPES USING LONG-READ SINGLE MOLECULE, REAL-TIME SEQUENCING OF FOSMIDS

Aim To implement an automated workflow for the assembly of complex immunogenetic regions using pooled fosmids and SMRT® Sequencing. Methods Here we present a simple automated workflow for the assembly of complete haplotypes from pools of tiled fosmids using a single library preparation and sequencing run with a quick turnaround time using an automated pipeline. The long reads generated in SMRT® Sequencing together with the hierarchical genome assembly process (HGAP) algorithm make it possible to fully assemble data with high accuracy even when the underlying structure is highly repetitive. Long reads are first preassembled into highly accurate error corrected reads before an overlap layout consensus (OLC) assembly to generate complete fosmid sequences. Fosmid sequences are trimmed of any vector before being overlapped to from a continuous haplotype. Results We show complete assembly of ∼150–200 kb haplotypes of the KIR locus, characterized by a large number of tandem repeats, from a single sequencing run. The assembly has a high base accuracy when compared to known references. Discussion The ability to haplotype and sequence complex immunogenetic regions will facilitate the integration of long-range LD in Conserved Extended Haplotypes in the MHC region and LRC region. Local fine-scale LD data will bring exciting opportunities to explore the evolution of disease associations of the immune sub-genome. A tiling of targeted fosmids can be used to clone extended lengths of genomic DNA, 100s of kb in length, but repeat complexity in regions of particular interest, such as the major histocompatibility complex (MHC) and the killer Ig-like receptor (KIR) locus, means that sequence assembly of complete haplotypes is difficult and often requires expert knowledge. This simple and robust assembly approach can be scaled-up allowing a complex genomic region to be sequenced in a population genetics setup. Such sequencing would be of high value in disease association research. R.J. Hall: Employee; Company/Organization; Pacific Biosciences. K. Eng: Employee; Company/Organization; Pacific Biosciences. L. Hon: Employee; Company/Organization; Pacific Biosciences. D.E. Geraghty: Employee; Company/Organization; Scisco Genetics Inc. S. Ranade: Employee; Company/Organization; Pacific Biosciences.

Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data

We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.