Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies.

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent ('Rapid' protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel 'Rapid' method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the 'Rapid' protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel 'Rapid' DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel 'Rapid' DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing.

Whole-genome sequencing reveals a link between ß-lactam resistance and synthetases of the alarmone (p)ppGpp in Staphylococcus aureus.

The overwhelming majority of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates exhibit a peculiar heterogeneous resistance to ß-lactam antibiotics: in cultures of such strains, the majority of cells display only a low level of methicillin resistance--often close to the MIC breakpoint of susceptible strains. Yet, in the same cultures, subpopulations of bacteria exhibiting very high levels of resistance are also present with variable frequencies, which are characteristic of the particular MRSA lineage. The mechanism of heterogeneous resistance is not understood. We describe here an experimental system for exploring the mechanism of heterogeneous resistance. Copies of the resistance gene mecA cloned into a temperature-sensitive plasmid were introduced into the fully sequenced methicillin-susceptible clinical isolate S. aureus strain 476. Transductants of strain 476 expressed methicillin resistance in a heterogeneous fashion: the great majority of cells showed only low MIC (0.75 µg/ml) for the antibiotic, but a minority population of highly resistant bacteria (MIC >300 µg/ml) was also present with a frequency of ∼10(-4). The genetic backgrounds of the majority and minority cells were compared by whole-genome sequencing: the only differences detectable were two point mutations in relA of the highly resistant minority population of bacteria. The relA gene codes for the synthesis of (p)ppGpp, an effector of the stringent stress response. Titration of (p)ppGpp showed increased amounts of this effector in the highly resistant cells. Involvement of (p)ppGpp synthesis genes may explain some of the perplexing aspects of ß-lactam resistance in MRSA, since many environmental and genetic changes can modulate cellular levels of (p)ppGpp.

Our second genome-human metagenome: how next-generation sequencer changes our life through microbiology.

Next-generation sequencing has greatly expanded our ability to query the identity and genetic composition of entire communities of microbial organisms. This area of research, known as metagenomics, does not rely upon culturing the individual organisms. Rather, the genetic material from the entire community is processed and sequenced simultaneously. From this sequence data, researchers are able to determine the relative population of organisms within the community as well as determine which genes and metabolic pathways are present and expressed in the microbial community. While these techniques have been applied to a wide range of environmental samples, metagenomics is also the focus of intensive research on human-associated microbial communities. The scope of these human metagenomics studies are quite varied, but all have a common goal of attempting to understand the important role that human commensal microbial communities play in health and disease. The early results from studying the human metagenome indicate a vital role that microbial communities play in immunity, health, and disease. Going forward, human metagenomics is a wide open field of research with many unanswered questions such as which factors are responsible for the variation of composition of an individual's microbiome, how does the microbiome respond to disturbance, and what beneficial functions are the microorganisms performing?

Comprehensive resequence analysis of a 136 kb region of human chromosome 8q24 associated with prostate and colon cancers.

Recently, genome-wide association studies have identified loci across a segment of chromosome 8q24 (128,100,000-128,700,000) associated with the risk of breast, colon and prostate cancers. At least three regions of 8q24 have been independently associated with prostate cancer risk; the most centromeric of which appears to be population specific. Haplotypes in two contiguous but independent loci, marked by rs6983267 and rs1447295, have been identified in the Cancer Genetic Markers of Susceptibility project ( http://cgems.cancer.gov ), which genotyped more than 5,000 prostate cancer cases and 5,000 controls of European origin. The rs6983267 locus is also strongly associated with colorectal cancer. To ascertain a comprehensive catalog of common single-nucleotide polymorphisms (SNPs) across the two regions, we conducted a resequence analysis of 136 kb (chr8: 128,473,000-128,609,802) using the Roche/454 next-generation sequencing technology in 39 prostate cancer cases and 40 controls of European origin. We have characterized a comprehensive catalog of common (MAF > 1%) SNPs within this region, including 442 novel SNPs and have determined the pattern of linkage disequilibrium across the region. Our study has generated a detailed map of genetic variation across the region, which should be useful for choosing SNPs for fine mapping of association signals in 8q24 and investigations of the functional consequences of select common variants.

Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome.

BACKGROUND: With a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon (Salmo salar) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering approximately 1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library. RESULTS: An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with approximately 30x coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (approximately 0.09x coverage) were incorporated. The addition of paired end sequencing reads (additional approximately 26x coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (approximately 10.5x coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly. CONCLUSION: These results represent the first use of GS FLX paired end reads for de novo sequence assembly. Our data demonstrated that this improved the GS FLX assemblies; however, with respect to de novo sequencing of complex genomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently, for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sanger technology.

Unique features of a highly pathogenic Campylobacter jejuni strain.

Campylobacter jejuni, a major human enteric pathogen, exhibits significant strain-to-strain differences which result in differences in pathogenic potential. C. jejuni 81-176 is a highly virulent strain that exhibits unique pathogenic features and is used by many research laboratories. We have determined the nucleotide sequence of its genome and compared it to the genomes of other sequenced C. jejuni strains. We identified a number of unique genetic features which may confer specific metabolic and pathogenic properties on this strain. We have also identified regions of the C. jejuni genome that are hot spots for the integration of horizontally acquired genetic material. This information should help the understanding of the pathogenesis of C. jejuni and, in particular, the unique features of this highly pathogenic strain.

Genome sequencing in microfabricated high-density picolitre reactors.

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Next generation sequencing technologies.

From the investigation of disease-associated loci in humans, to monitoring the changing genomes of pathogenic viruses and bacteria, sequencing is a powerful and versatile tool. A new generation of sequencing technologies will increase the speed and lower the cost of sequencing, and promises to expand the utility of sequencing in drug discovery and development.: