Whole Genome Sequencing Methods Research Articles

The Effect of Nucleic Acid Extraction Platforms and Sample Storage on the Integrity of Viral RNA for Use in Whole Genome Sequencing

Extraction of viral RNA and the storage of sample material are extremely important factors in the detection and whole genome sequencing (WGS) of viral pathogens. Although PCR-based detection methods focus on small amplicons, viral WGS applications require RNA of high quality and integrity for adequate sequence coverage and depth. This study examined the fitness of one manual and four automated RNA extraction platforms commonly used in diagnostic laboratories for use in metagenomic sequencing, how the practice of storing sample material in Qiagen buffer AVL before extraction affected the integrity of viral RNA and its suitability for use in amplicon-based WGS methods, and how the addition of Triton X-100 to buffer AVL affected the capability of the extraction platforms and the integrity of viral RNA in stored samples. This study found that the EZ1 platform gave the best performance of the automated platforms and gave comparable results to the frequently used manual Qiagen extraction protocol when extracted viral RNA was used in metagenomics sequencing. To maintain high levels of viral RNA integrity suitable for amplicon-based WGS, nucleic acid should be extracted from samples immediately, because even short storage periods in buffer AVL have a severe effect on integrity, and the addition of Triton X-100 had little effect on the quality of viral material for WGS.

MOST: a modified MLST typing tool based on short read sequencing.

Multilocus sequence typing (MLST) is an effective method to describe bacterial populations. Conventionally, MLST involves Polymerase Chain Reaction (PCR) amplification of housekeeping genes followed by Sanger DNA sequencing. Public Health England (PHE) is in the process of replacing the conventional MLST methodology with a method based on short read sequence data derived from Whole Genome Sequencing (WGS). This paper reports the comparison of the reliability of MLST results derived from WGS data, comparing mapping and assembly-based approaches to conventional methods using 323 bacterial genomes of diverse species. The sensitivity of the two WGS based methods were further investigated with 26 mixed and 29 low coverage genomic data sets from Salmonella enteridis and Streptococcus pneumoniae. Of the 323 samples, 92.9% (n = 300), 97.5% (n = 315) and 99.7% (n = 322) full MLST profiles were derived by the conventional method, assembly- and mapping-based approaches, respectively. The concordance between samples that were typed by conventional (92.9%) and both WGS methods was 100%. From the 55 mixed and low coverage genomes, 89.1% (n = 49) and 67.3% (n = 37) full MLST profiles were derived from the mapping and assembly based approaches, respectively. In conclusion, deriving MLST from WGS data is more sensitive than the conventional method. When comparing WGS based methods, the mapping based approach was the most sensitive. In addition, the mapping based approach described here derives quality metrics, which are difficult to determine quantitatively using conventional and WGS-assembly based approaches.

Absence of genomic hypomethylation or regulation of cytosine-modifying enzymes with aging in male and female mice.

BackgroundChanges to the epigenome with aging, and DNA modifications in particular, have been proposed as a central regulator of the aging process, a predictor of mortality, and a contributor to the pathogenesis of age-related diseases. In the central nervous system, control of learning and memory, neurogenesis, and plasticity require changes in cytosine methylation and hydroxymethylation. Although genome-wide decreases in methylation with aging are often reported as scientific dogma, primary research reports describe decreases, increases, or lack of change in methylation and hydroxymethylation and their principle regulators, DNA methyltransferases and ten-eleven translocation dioxygenases in the hippocampus. Furthermore, existing data are limited to only male animals.ResultsThrough examination of the hippocampus in young, adult, and old male and female mice by antibody-based, pyrosequencing, and whole-genome oxidative bisulfite sequencing methods, we provide compelling evidence that contradicts the genomic hypomethylation theory of aging. We also demonstrate that expression of DNA methyltransferases and ten-eleven translocation dioxygenases is not differentially regulated with aging or between the sexes, including the proposed cognitive aging regulator DNMT3a2. Using oxidative bisulfite sequencing that discriminates methylation from hydroxymethylation and by cytosine (CG and non-CG) context, we observe sex differences in average CG methylation and hydroxymethylation of the X chromosome, and small age-related differences in hydroxymethylation of CG island shores and shelves, and methylation of promoter regions.ConclusionThese findings clarify a long-standing misconception of the epigenomic response to aging and demonstrate the need for studies of base-specific methylation and hydroxymethylation with aging in both sexes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0080-6) contains supplementary material, which is available to authorized users.

PWE-102 Elucidating The Role of Non JEJUNI/Coli-Campylobacter in The Development of Colorectal Cancer Utilising Comparative Genomics to Study Their Pathogenic Potential

Introduction Emerging evidence links the gut microbiota to colonic inflammation and colorectal cancer (CRC). Studies have shown over-representation of oral bacterial species including Campylobacter spp. in CRC tissue and also njc-Campylobacter are over-represented in IBD wherein normal homeostasis has been lost. Understanding the role of njc-Campylobacter as an aetiological agent in CRC and elucidating their pathogenicity characteristics is of immense clinical importance. Aim To perform whole genome sequencing (WGS) of clinically derived njc-Campylobacter to compare/identify virulence traits potentially linked to disease presentation. Methods WGS of 21 unique clinically derived Campylobacter isolates was performed (7 Campylobacter showae, 3 Campylobacter ureolyticus and 11 Campylobacter concisus) from biopsy/faecal samples of subjects with malignancy, acute enteritis and IBD. DNA was extracted using the QIAamp DNA Mini Kit. Libraries were prepared using Illumina Nextera XT DNA Library Prep Kit and Nextera XT Index Kit v2, multiplexed, and paired-end sequenced on the Illumina MiSeq. De novo read assembly used the A5-miseq pipeline with QUAST. Genome annotation was performed using the rapid prokaryotic genome annotation tool Prokka. Results Genome assembly of C. showae strains resulted in 8 to 59 contigs per strain (mean number = 21; average genome size 2,199,446 nucleotides); C. concisus isolates had an average contig number of 72 and average genome size of 1,982,586 nucleotides. C. ureolyticus strains average contig number was 134 and average genome size of 1,608,008 nucleotides. Through Prokka annotation, the average number of coding sequences was 2215 for C. showae isolates (range 2050–2368) with function predicted for ~71.3%. For C. concisus the average number of coding sequences was 1985 (range 1833–2087) with function predicted for ~ 71.9%. C. ureolyticus had a lower number of coding sequences (average 1613) with function predicted for ~72.6%. We focused on the VirB4/D10 operon homologs, which are components of the Type IV secretion system. Contrary to previously published sequence data from oral strains, the majority of colonic C. showae isolated and 2 C. concisus strains possessed almost complete T4SS systems and additional virulence traits, including the exotoxin 9 gene. Conclusion The findings clearly demonstrate genetic differences among colonic Campylobacter isolates as compared to oral strains and sheds light on the pathogenic potential of these emerging colonic pathogens. The findings also further highlight the need for further sequencing studies to be undertaken. Disclosure of Interest None Declared

Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation.

Epigenetic regulation through DNA methylation (5mC) plays an important role in development, aging, and a variety of diseases. Genome-wide studies of base- and strand-specific 5mC are limited by the extensive sequencing required. Targeting bisulfite sequencing to specific genomic regions through sequence capture with complimentary oligonucleotide probes retains the advantages of bisulfite sequencing while focusing sequencing reads on regions of interest, enables analysis of more samples by decreasing the amount of sequence required per sample, and provides base- and strand-specific absolute quantitation of CG and non-CG methylation levels. As an example, an oligonucleotide capture set to interrogate 5mC levels in all rat RefSeq gene promoter regions (18,814) and CG islands, shores, and shelves (18,411) was generated. Validation using whole-genome methylation standards and biological samples demonstrates enrichment of the targeted regions and accurate base-specific quantitation of CG and non-CG methylation for both forward and reverse genomic strands. A total of 170Mb of the rat genome is covered including 6.6 million CGs and over 67 million non-CG sites, while reducing the amount of sequencing required by ~85% as compared to existing whole-genome sequencing methods. This oligonucleotide capture targeting approach and quantitative validation workflow can also be applied to any genome of interest.

A fast and robust method for whole genome sequencing of the Aleutian Mink Disease Virus (AMDV) genome

Aleutian Mink Disease Virus (AMDV) is a frequently encountered pathogen associated with commercial mink breeding. AMDV infection leads to increased mortality and compromised animal health and welfare. Currently little is known about the molecular evolution of the virus, and the few existing studies have focused on limited regions of the viral genome.This paper describes a robust, reliable, and fast protocol for amplification of the full AMDV genome using long-range PCR. The method was used to generate next generation sequencing data for the non-virulent cell-culture adapted AMDV-G strain as well as for the virulent AMDV-Utah strain. Comparisons at nucleotide- and amino acid level showed that, in agreement with existing literature, the highest variability between the two virus strains was found in the left open reading frame, which encodes the non-structural (NS1–3) genes. This paper also reports a number of differences that potentially can be linked to virulence and host range.To the authors’ knowledge, this is the first study to apply next generation sequencing on the entire AMDV genome. The results from the study will facilitate the development of new diagnostic tools and can form the basis for more detailed molecular epidemiological analyses of the virus.

TLR21's agonists in combination with Aeromonas antigens synergistically up-regulate functional TLR21 and cytokine gene expression in yellowtail leucocytes

The purpose of this study was to characterize the TLR21 gene from yellowtail (Seriola lalandi) and its functional activity using TLR agonist stimulation and Aeromonas antigens. The TLR21 nucleotide sequence from yellowtail was obtained using the whole-genome shotgun sequencing method and bioinformatics tools. Basal TLR21 gene expression was analyzed in several tissues. Subsequently, the gene expression of TLR21 and cytokines IL-1β and TNF-α was evaluated in TLR agonist (CpG-ODN2006, LPS, and Poly I:C) exposing head kidney leucocytes, which were then subjected to Aeromonas antigen stimulation. The yellowtail full-length cDNA sequence of SlTLR21 was 3615 bp (980 aa) showing a high degree of similarity with the counterparts of other fish species and sharing the common structural architecture of the TLR family, including LRR domains, one C-terminal LRR region, and a TIR domain. Gene expression studies revealed the constitutive expression of TLR21 mRNA in all the analyzed tissues; the highest levels were observed in spleen and head kidney where they play an important role in the fish immune system. Transcripts of TLR21 and the downstream IL-1β and TNF-α cytokine genes were most strongly up-regulated after exposure to the TLR agonists following Aeromonas antigen stimulation, suggesting they are involved in immune response. The results indicated that TLR agonists, in combination with Aeromonas antigens in head kidney leucocytes, synergistically enhance TLR21 and cytokines in yellowtail.

Next generation sequencing for whole genome analysis and surveillance of influenza A viruses

BackgroundThe Wadsworth Center, New York State Department of Health (NYSDOH), conducts routine diagnosis and surveillance of influenza viruses. Whole genome sequencing (WGS) with next generation sequencing (NGS) was initiated to provide more rapid, detailed, thorough, and accurate analysis. ObjectivesTo optimize and implement a method for routine WGS of influenza A viruses. To use WGS to monitor influenza A viruses for reassortment, mutations associated with antiviral resistance and antigenicity changes, as well as those potentially affecting virulence and tropism. Study designMultiple extraction and amplification methods were investigated and optimized for the production of template to be used for NGS. Additionally, software options were considered for data analysis. Initial WGS influenza projects have included the comparison of mixed population sequence data obtained with NGS, Sanger dideoxy sequencing, and pyrosequencing, the comparison of sequences obtained from paired primary/cultured samples, the analysis of sequence changes over several influenza seasons, and phylogenetic analysis. ResultsProcedures were optimized for extraction and amplification such that WGS could be successfully performed on both cultured isolates and primary specimens. Data is presented on 15 A/H1pdm09 and 44 A/H3N2 samples. Analysis of influenza A viruses identified and confirmed variant and mixed populations affecting antigenicity and antiviral susceptibility in both primary specimens and cultured isolates. ConclusionsAn influenza A whole genome PCR method has been optimized for the reliable production of template for NGS. The WGS method has been successfully implemented for enhanced comprehensive surveillance and the generation of detailed clinical data on drug resistance and virulence. Data obtained with this method will also aid in future vaccine selection.

Whole Genome Sequencing Increases Molecular Diagnostic Yield Compared with Current Diagnostic Testing for Inherited Retinal Disease

PurposeTo compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD).DesignCase series.ParticipantsA total of 562 patients diagnosed with IRD.MethodsWe performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data.Main Outcome MeasuresDiagnostic yield of genomic testing.ResultsAcross known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15–45) uplift in diagnostic yield.ConclusionsWe show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS.

Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

ObjectiveTo evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform. MethodsVirus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. ResultsThe M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus. ConclusionThe M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

Novel genomic microsatellite markers for genetic population and diversity studies of tropical scalloped spiny lobster (Panulirus homarus) and their potential application in related Panulirus species.

Fourteen polymorphic microsatellites with perfect di-, tri-, and tetra-nucleotide repeats were identified for Panulirus homarus using Roche 454 whole-genome sequencing method. Microsatellites were efficiently co-amplified in four multiplexes and a singleplex, providing consistent and easily interpretable genotypes. The number of alleles per locus ranged from 2 to 11 with the observed and expected heterozygosity ranging between 0.000-0.532 and 0.031-0.836, respectively. A significant deviation from Hardy-Weinberg equilibrium was observed for majority of the loci, probably due to homozygote excess. Genetic linkage disequilibrium analysis between all the possible pairs of the loci showed significant departure from the null hypothesis in the loci pairs Pho-G11-Pho-G33 and Pho-G33-Pho-G57. High success in primer cross-species amplification of these microsatellite markers indicates their utility for genetic studies of different Panulirus species.

Helicobacter pylori adaptation in vivo in response to a high-salt diet.

Helicobacter pylori exhibits a high level of intraspecies genetic diversity. In this study, we investigated whether the diversification of H. pylori is influenced by the composition of the diet. Specifically, we investigated the effect of a high-salt diet (a known risk factor for gastric adenocarcinoma) on H. pylori diversification within a host. We analyzed H. pylori strains isolated from Mongolian gerbils fed either a high-salt diet or a regular diet for 4 months by proteomic and whole-genome sequencing methods. Compared to the input strain and output strains from animals fed a regular diet, the output strains from animals fed a high-salt diet produced higher levels of proteins involved in iron acquisition and oxidative-stress resistance. Several of these changes were attributable to a nonsynonymous mutation in fur (fur-R88H). Further experiments indicated that this mutation conferred increased resistance to high-salt conditions and oxidative stress. We propose a model in which a high-salt diet leads to high levels of gastric inflammation and associated oxidative stress in H. pylori-infected animals and that these conditions, along with the high intraluminal concentrations of sodium chloride, lead to selection of H. pylori strains that are most fit for growth in this environment.

A 26-hour system of highly sensitive whole genome sequencing for emergency management of genetic diseases.

While the cost of whole genome sequencing (WGS) is approaching the realm of routine medical tests, it remains too tardy to help guide the management of many acute medical conditions. Rapid WGS is imperative in light of growing evidence of its utility in acute care, such as in diagnosis of genetic diseases in very ill infants, and genotype-guided choice of chemotherapy at cancer relapse. In such situations, delayed, empiric, or phenotype-based clinical decisions may meet with substantial morbidity or mortality. We previously described a rapid WGS method, STATseq, with a sensitivity of >96 % for nucleotide variants that allowed a provisional diagnosis of a genetic disease in 50 h. Here improvements in sequencing run time, read alignment, and variant calling are described that enable 26-h time to provisional molecular diagnosis with >99.5 % sensitivity and specificity of genotypes. STATseq appears to be an appropriate strategy for acutely ill patients with potentially actionable genetic diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0221-8) contains supplementary material, which is available to authorized users.

The Sequence of the Human Genome

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies—a whole-genome assembly and a regional chromosome assembly—were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional ∼12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

Deriving HLA genotyping from whole genome sequencing data using Omixon HLA twin(tm) in G3’s global clinical study

Aim Identify the degree of concordance and the ambiguities when DNA samples are analyzed using two HLA genotyping systems; Whole Genome Sequencing (WGS) data analyzed by Omixon HLA Target software and genotyping by the Holotype HLA typing system. Methods WGS data were generated of 235 human samples from G3’s GLOBAL study (NCT01738828) using paired 100 bps long HiSeq Illumina ∼30× coverage and analyzed by the Omixon HLA Target software. Genotyping was also performed for the same samples using the Holotype HLA typing system, whereby the generated libraries were sequenced on the Illumina MiSeq using the 250 bps paired-end sequencing protocol. Data were analyzed by the Omixon HLA Twin software. Concordance at the three-field level between the two approaches was assessed for each of the HLA-A, B, C, DQB1 and DRB1 loci. Ambiguities were also determined for each of the typing methods. Results A pairwise comparison between the two NGS-based methods shows that in average they were 97.2% concordant at the three-fields level (HLA-A: 450/470 95.7%, HLA-B: 464/470 98.7%, HLA-C: 464/470 98.7%, HLA-DQB1: 438/470 93.2%, HLA-DRB1 467/468 99.8%). Most of the discrepancies were due to allele dropout in the WGS sample or systematic errors of the software. The 41 non-systematic mistypings were due to either insufficient coverage of the WGS data or algorithmic problems. The Holotype HLA system did not generate any ambiguities for the HLA-A, -B, -C and HLA-DQB1 loci at the three-fields typing level. However, as intron 1 and exon 1 of DRB1 are not targeted, 1.4% of ambiguities were generated for the DRB1 locus.The WGS typing considers only the exons of the reference database, leading to phase ambiguities and overall ambigous typings of 9.8%, 4.3%, 7.7%, 8.3% and 3.4% for HLA-A, B, C, DQB1 and DRB1 respectively. Conclusions HLA genotyping using WGS data derived through next-generation sequencing and analyzed by the Omixon HLA Twin software is possible at 97.2% accuracy level. Future improvements of the software addressing ambiguities and remaining systematic errors can make HLA typing from WGS data more reliable. S. Juhos: Employee; Company/Organization; Omixon Ltd. T. V a g o : Employee; Company/Organization; Omixon Ltd. D. Ferriola: Employee; Company/Organization; Children’s Hospital of Philadelphia. J. Duke: Employee; Company/Organization; Children’s Hospital of Philadelphia. S. V o r o s: Employee; Company/Organization; Global Genomics Group. B.O. Brown: Employee; Company/Organization; Global Genomics Group. I. Marvasty: Employee; Company/Organization; Global Genomics Group. T. Hague: Employee; Company/Organization; Omixon Ltd. D. Monos: Employee; Company/Organization; Children’s Hospital of Philadelphia.

Whole-genome sequencing for national surveillance of Shiga toxin-producing Escherichia coli O157.

National surveillance of gastrointestinal pathogens, such as Shiga toxin-producing Escherichia coli O157 (STEC O157), is key to rapidly identifying linked cases in the distributed food network to facilitate public health interventions. In this study, we used whole-genome sequencing (WGS) as a tool to inform national surveillance of STEC O157 in terms of identifying linked cases and clusters and guiding epidemiological investigation. We retrospectively analyzed 334 isolates randomly sampled from 1002 strains of STEC O157 received by the Gastrointestinal Bacteria Reference Unit at Public Health England, Colindale, in 2012. The genetic distance between each isolate, as estimated by WGS, was calculated and phylogenetic methods were used to place strains in an evolutionary context. Estimates of linked clusters representing STEC O157 outbreaks in England and Wales increased by 2-fold when WGS was used instead of traditional typing techniques. The previously unidentified clusters were often widely geographically distributed and small in size. Phylogenetic analysis facilitated identification of temporally distinct cases sharing common exposures and delineating those that shared epidemiological and temporal links. Comparison with multi locus variable number tandem repeat analysis (MLVA) showed that although MLVA is as sensitive as WGS, WGS provides a more timely resolution to outbreak clustering. WGS has come of age as a molecular typing tool to inform national surveillance of STEC O157; it can be used in real time to provide the highest strain-level resolution for outbreak investigation. WGS allows linked cases to be identified with unprecedented specificity and sensitivity that will facilitate targeted and appropriate public health investigations.

SISPA-Seq for rapid whole genome surveys of bacterial isolates

Whole genome sequencing (WGS) of large isolate collections has many applications, yet sequencing costs are still significant. We sought to develop a rapid and cost efficient WGS method to address fundamental questions in clinical microbiology. We evaluated the performance of SISPA (Sequence-Independent, Single-Primer Amplification) combined with next-generation sequencing (SISPA-Seq) of 75 clinical isolates of Acinetobacter baumannii to establish whether SISPA-Seq resulted in sufficient coverage and quality to (1) determine strain phylogenetic placement and (2) and carriage of known antibiotic resistance (AbR) genes. Strains for which whole genome sequences were available were included for validation. Two libraries for each strain were constructed from separate SISPA reactions with different barcoded primers, using genomic DNA prepared from either high quality or rapid heat-lysis preparations. SISPA-Seq resulted in a median of 65× genome coverage when reads from both primer sets were combined. Coverage and quality were sufficient for detection of AbR genes by comparison of reads to the ARG-ANNOT database and were often sufficient to distinguish between different allelic variants of the same gene. kSNP and RAxML were used to construct a robust phylogeny based on single-nucleotide variants (SNVs) that showed that the SISPA-Seq data was sufficient for sensitive and accurate phylogenetic placement. Advantages of the SISPA-Seq method include inexpensive and rapid DNA preparation and a typical total cost less than one-half that of standard genome sequencing. In summary, SISPA-Seq can be used to survey whole genomes of a large strain collection and identify strains that should be targeted for additional sequencing.

Improved tagmentation-based whole-genome bisulfite sequencing for input DNA from less than 100 mammalian cells.

To develop a whole-genome methylation sequencing method that fulfills the needs for studies using ultra-low-input DNA. The tagmentation-based whole-genome bisulfite sequencing (T-WGBS) technology is modified, enabling stable library construction with complexity from minimally 0.5 ng of initial genomic DNA, which equals less than 100 mammalian cells. We thoroughly assessed the performance of this T-WGBS method by sequencing the methylomes of a rice strain and pre-implantation embryos of rhesus monkey and compare to traditional WGBS approach, thereby demonstrating the efficacy of this new approach. This new approach is highly attractive for the complete methylome analysis of very few cells, for example, mammalian pre-implantation embryos, or tiny human biopsy specimens.

Characterization of genomic variations in SNPs of PE_PGRS genes reveals deletions and insertions in extensively drug resistant (XDR) M. tuberculosis strains from Pakistan

BackgroundMycobacterium tuberculosis (MTB) PE_PGRS genes belong to the PE multigene family. Although the function of PE_PGRS genes is unknown, it is hypothesized that the PE_PGRS genes may be associated with antigenic variability in MTB. Material and methodsWhole genome sequencing analysis was performed on (n=37) extensively drug-resistant (XDR) MTB strains from Pakistan, which included Lineage 1 (East African Indian, n=2); Other lineage 1 (n=3); Lineage 3 (Central Asian, n=24); Other lineage 3 (n=4); Lineage 4 (X3, n=1) and T group (n=3) MTB strains. ResultsThere were 107 SNPs identified from the analysis of 42 PE_PGRS genes; of these, 13 were non-synonymous SNPs (nsSNPs). The nsSNPs identified in PE_PGRS genes – 6, 9 and 10 – were common in all EAI, CAS, Other lineages (1 and 3), T1 and X3. Deletions (DELs) in PE_PGRS genes – 3 and 19 – were observed in 17 (80.9%) CAS1 and 6 (85.7%) in Other lineages (1 and 3) XDR MTB strains, while DELs in the PE_PGRS49 were observed in all CAS1, CAS, CAS2 and Other lineages (1 and 3) XDR MTB strains. All CAS, EAI and Other lineages (1 and 3) strains showed insertions (INS) in PE_PGRS6 gene, while INS in the PE_PGRS genes 19 and 33 were observed in 20 (95.2%) CAS1, all CAS, CAS2, EAI and Other lineages (1 and 3) XDR MTB strains. ConclusionGenetic diversity in PE_PGRS genes contributes to antigenic variability and may result in increased immunogenicity of strains. This is the first study identifying variations in nsSNPs and INDELs in the PE_PGRS genes of XDR-TB strains from Pakistan. It highlights common genetic variations which may contribute to persistence.

Analysis of a long-term outbreak of XDR Pseudomonas aeruginosa: a molecular epidemiological study.

Here we report on a long-term outbreak from 2009 to 2012 with an XDR Pseudomonas aeruginosa on two wards at a university hospital in southern Germany. Whole-genome sequencing was performed on the outbreak isolates and a core genome was constructed for molecular epidemiological analysis. We applied a time-place-sequence algorithm to improve estimation of transmission probabilities. By using conventional infection control methods we identified 49 P. aeruginosa strains, including eight environmental isolates that belonged to ST308 (by MLST) and carried the metallo-β-lactamase IMP-8. Phylogenetic analysis on the basis of a non-recombinant core genome that contained 22 outbreak-specific SNPs revealed a pattern of four dominant clades with a strong phylogeographic structure and allowed us to determine the potential temporal origin of the outbreak to July 2008, 1 year before the index case was diagnosed. Superspreaders at the root of clades exhibited a high number of probable and predicted transmissions, indicating their exceptional position in the outbreak. Our results suggest that the initial expansion of dominant sublineages was driven by a few superspreaders, while environmental contamination seemed to sustain the outbreak for a long period despite regular environmental control measures.