High throughput sequencing in veterinary infection biology

High Throughput Sequencing In Veterinary Infection Biology-Free PDF

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894 Rev sci tech Off int Epiz 32 3, are now providing more powerful diagnostic opportunities de novo assembly of novel microorganisms 14 15 16 In. as well as improved classification forecasting and therapy addition to having the longest read lengths which simplifies. selection for many infectious illnesses de novo assembly this was also the first NGS platform on the. market becoming available in 2005 In 2007 the 454 FLX. The importance of nucleotide sequencing was fully realised system was followed by the Genome Analyser developed by. after the establishment of the Sanger method 5 For the past Illumina Solexa 17. 35 years this technology has been the dominant approach. for DNA sequencing It enabled the first viral genomes. to be sequenced 6 and also laid the foundation for the The field has since developed rapidly as a result of the. development of polymerase chain reaction PCR which continuous improvement and refinement of existing. is the best known and most successfully implemented systems and the release of completely new platforms such. diagnostic molecular technology to date 7 8 as the Ion Torrent Personal Genome Machine PGM As. a consequence the efficiency and throughput of DNA. Since the first versions of the Sanger techniques sequencing sequencing are now increasing at a rate even faster than. methods have rapidly improved generating very powerful that projected by Moore s law for computing power a. tools for detecting and identifying various pathogens such doubling every two years 18 19 This has also resulted. as viruses bacteria and other microbes An important step. in a dramatic reduction in DNA sequencing costs making. in this development was the introduction of automated. the technology more accessible to the average laboratory. multicapillary based instruments using fluorophore labelling. with multi spectral imaging later referred to as first generation Fig 1. sequencing platforms 9 This type of Sanger sequencing is. still extensively used in laboratories around the world and is a Recently a new generation of single molecule sequencing. front line diagnostic tool for virus characterisation including technologies termed third generation sequencing TGS. pathotyping and phylogenetic analysis has emerged as summarised for example in the review. article of Schadt et al 20, The OIE has actively supported the early adoption and use. of these molecular techniques in the field of veterinary In this White Paper of the OIE several approaches and. medicine through the OIE Reference Laboratories and by the. examples of high throughput sequencing HTS are, establishment of OIE Collaborating Centres with a strong focus. on biotechnology based diagnosis in veterinary medicine summarised and their diagnostic applicability is discussed. briefly Selected future aspects of HTS are also outlined with. The first generation sequencing approaches have opened a focus on improved diagnosis and control of infectious. up new pathways for the detection and identification diseases in veterinary medicine. of various pathogens host pathogen interactions and. the evolution of infectious agents However attempts to. sequence larger genomes such as the whole genomes of. various animal species using multicapillary sequencing 10 000. have encountered considerable bottlenecks in throughput. scalability speed and resolution This has spurred the 1 000. Cost US dollars per MB, development of new sequencing technologies 10 11 Moore s Law. The subsequent major technological advances in cyclic array. sequencing gave rise to what is known as second generation 10. sequencing SGS or next generation sequencing NGS, These technologies involve iterative cycles during which the 1.
sequences of DNA features which have been immobilised. to constant locations on a solid substrate are determined. one base position at a time using enzymatic manipulation. and imaging based data collection 12 13 Today there are. several different NGS platforms with tailored protocols and 0. 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012. approaches to template preparation and sequencing that. determine the type of data produced Fig 1, The change over time for cost per raw megabase of DNA. The 454 FLX pyrosequencing platform 13 was established sequencing. as the high throughput method of choice for discovery and Source www genome gov sequencingcosts. No 04122013 20 EN,Rev sci tech Off int Epiz 32 3 895. High throughput sequencing Sample preparation,Most sample preparation and enrichment protocols. as a diagnostic tool are composed of several individual steps including. homogenisation filtration ultracentrifugation and nuclease. As demonstrated by several peer reviewed articles HTS treatment as well as nucleic acid extraction and purification. has shown great potential in the detection and discovery followed by amplification These steps are outlined briefly. of novel pathogens see Examples of the application of below. high throughput sequencing in veterinary diagnostic. microbiology below In this regard it is common to Depending on the nature of the starting material i e type of. distinguish between the spread of known infections to new tissue blood or other body fluids it may first be necessary. areas and or the emergence of completely novel unknown to homogenise fractionise or dilute a representative amount. pathogens Contrary to earlier techniques HTS is unbiased of the original sample This can be performed according to. and reports all nucleotide sequences present in the original existing protocols for other purposes or applications and it is. sample However as with earlier techniques the lower important to remember that the choice of procedure can affect. limit for detection is still ultimately determined by the the final outcome due to differences in host cell disruption. abundance of pathogens in relation to host background and preservation of intact pathogens 22 It is therefore. material By enabling deeper sequencing to be performed advisable to find a homogenisation procedure optimised. more quickly and cheaply the continuing development of for each sample matrix rather than a one fits all solution. HTS techniques is also continuing to improve the likelihood 26 Regardless of the initial procedure centrifugation of. of detecting low copy number pathogens 21 In addition the homogenates and fluids followed by microfiltration is an. sampling sample preparation and enrichment protocols effective method to separate free and released pathogens from. have all been demonstrated to have dramatic effects on the larger particles such as cellular debris. outcome of HTS based diagnostics 22 and thus should. each be considered as integral steps in the overall detection To purify and concentrate virus particles ultracentrifugation. scheme Fig 2 Since proper sampling of diagnostic has proven to be a reliable method especially in. specimens is a key issue for all laboratory investigations of combination with density gradients 27 28 Even though. an animal disease the subject has already been extensively a standard full sized ultracentrifuge is not readily available. covered in the OIE Manual of Diagnostic Tests and Vaccines in all laboratories smaller facilities might still consider a. for Terrestrial Animals and Manual of Diagnostic Tests for benchtop version if dealing with large quantities of small. Aquatic Animals 23 24 Besides the general rule that the volume samples. samples collected should be representative of the condition. being investigated downstream analysis and interpretation Nuclease treatment is a widely used enrichment method. rely on the inclusion of contextual metadata As argued by for viral nucleic acid and can be applied directly to. the Genomic Standards Consortium www gensc org with filtered material or as a complement to ultracentrifugation. sequencing costs for complete microbial and viral genomes Deoxyribonuclease DNase and ribonuclease RNase are. steadily decreasing the associated contextual information is used in combination or alone to remove host contaminants. increasingly valuable 25 by exploiting the protection from digestion afforded by. the virus capsid 29 30 Even though this is a powerful. method to remove the majority of host derived sequence. Clinical Sample preparation contaminants the material extracted after nuclease treatment. sampling enrichment, is often present in such small quantities that amplification is. required before sequencing Various sequence independent. Sequencing library amplification strategies have been employed and the two. construction, most widespread are phi29 polymerase based multiple.
displacement amplification 31 and random PCR using. Whole genome, sequencing modified versions of sequence independent single primer. amplification SISPA 30 32 Although a combined, Bioinformatics approach with nuclease treatment and amplification has. analyses been demonstrated to be very effective care should be. taken since nuclease treatment might destroy free microbial. Interpretation of results and viral nucleic acid if the samples are old or not well. preserved There is also a possibility that the amplification. Fig 2 method can introduce a bias 33 which is of less concern. Workflow for pathogen detection using high throughput for pathogen detection but may influence the recovery of. sequencing whole genome sequences,No 04122013 20 EN. 896 Rev sci tech Off int Epiz 32 3, For non culturable bacteria phi29 polymerase based 454 pyrosequencing Roche. amplification has been used successfully to obtain. sufficient quantities of DNA for sequencing 34 However The 454 pyrosequencing technology is based on emulsion. 16S ribosomal RNA rRNA gene deep sequencing has proven PCR emPCR for clonal amplification of single library. more useful than random amplification for metagenomics fragments on beads inside aqueous reaction bubbles in. studies of microbial communities In this approach primers combination with a sequencing by synthesis approach. designed for conserved regions in the bacterial 16S rRNA The DNA containing beads are loaded into individual wells. gene are fused with adaptors and barcodes and used to on a PicoTiterPlate which is subjected to a cyclic flow of. generate PCR products With the incorporation of adaptors sequentially added nucleotide reagents When a polymerase. and barcodes the products already constitute a sequencing mediated incorporation event occurs a chemiluminescent. library see Library construction and sequencing below enzyme generates an observable light signal that is recorded. and can be directly used for sequencing 35 The HTS of by a charge coupled device CCD camera 13 There. PCR products for specific loci is commonly referred to as are currently two platforms on the market using this. amplicon sequencing and can also be used as a powerful technology the GS FLX system and the GS Junior system. tool for the detection and identification of viruses and other While the latest GS FLX system can now generate around. microorganisms 36 700 megabases Mb of sequence data with read lengths of. up to 1 000 base pairs bp in a day it is still associated with. high running costs and is most commonly used by larger. Library construction and sequencing, sequencing facilities The GS Junior on the other hand is.
Most existing HTS platforms require the viral genomic essentially a smaller benchtop version with lower set up. sequences directly as DNA or reverse transcribed RNA and running costs aimed at research laboratories However. in the samples to be converted into sequencing libraries although 454 pyrosequencing has been the most commonly. suitable for subsequent cluster generation and sequencing used technology for HTS to date it has a high error rate. This process usually consists of four main steps in homopolymer regions i e three or more consecutive. identical DNA bases caused by accumulated light intensity. fragmentation of DNA performed by mechanical or variance 13. enzymatic shearing,SOLiD Life Technologies, end repair modification and ligation of adapters which. The sequencing by oligonucleotide ligation and detection. enable amplification of the sheared DNA by adapter specific. SOLiD technology also uses emPCR to generate clonal. DNA fragments on beads The enriched beads are then. deposited into separate pico wells on a glass slide to allow. size selection of DNA molecules with a certain optimal. sequencing by ligation This involves iterative rounds of. length for the current application or instrument, oligonucleotide ligation extension during which every. base is scored at least twice by using fluorescently labelled. enrichment of adapter ligated DNA by PCR 37 di or tri base probes with data translated into colour. space rather than conventional base space 12 While this. The last step is only necessary if the amount of starting approach provides very high accuracy the maximum read. material is small and has been observed to introduce length is relatively short 75 bp The SOLiD platforms have. amplification based artefacts 38 In addition by therefore been used mainly for applications not requiring de. adding specific short sequence tags indexes during the novo assembly of reads such as transcriptomics epigenomics. construction of multiple libraries it is possible to pool and resequencing of large mammalian genomes There are. libraries for increased throughput and reduced costs The currently two SOLiD platforms available the 5500 an. Corresponding author sandor belak slu se Summary Sequencing methods have improved rapidly since the first versions of the Sanger techniques facilitating the development of very powerful tools for detecting and identifying various pathogens such as viruses bacteria and other microbes

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