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structures in cancer revealed by ultra-deep sequencing.
The high mutation rate of RNA viruses enables the generation of a genetically diverse viral population, termed a quasispecies, within a single infected host. This high in-host genetic diversity enables an RNA virus to adapt to a diverse array of selective pressures such as host immune response and switching between host species. The negative-sense, single-stranded RNA virus, viral haemorrhagic septicaemia virus (VHSV), was originally considered an epidemic virus of cultured rainbow trout in Europe, but was later proved to be endemic among a range of marine fish species in the Northern hemisphere. To better understand the nature of a virus quasispecies related to the evolutionary potential of VHSV, a deep-sequencing protocol specific to VHSV was established and applied to 4 VHSV isolates, 2 originating from rainbow trout and 2 from Atlantic herring. Each isolate was subjected to Illumina paired end shotgun sequencing after PCR amplification and the 11.1 kb genome was successfully sequenced with an average coverage of 0.5–1.9 × 106 sequenced copies. Differences in single nucleotide polymorphism (SNP) frequency were detected both within and between isolates, possibly related to their stage of adaptation to host species and host immune reactions. The N, M, P and Nv genes appeared nearly fixed, while genetic variation in the G and L genes demonstrated presence of diverse genetic populations particularly in two isolates. The results demonstrate that deep sequencing and analysis methodologies can be useful for future in vivo host adaption studies of VHSV.
The results clearly demonstrated that the four isolates of VHSV were composed of a genetically diverse virus population, the first requirement for the formation of a quasispecies. Intra-host genetic variation has been demonstrated previously for other rhabdoviruses such as rabies virus  but to our knowledge, this study is the first to describe the intra-host genetic diversity in a novirhabdovirus using ultra deep sequencing.
31/08/2011 · Ultra-deep targeted sequencing ..
In general, resequencing projects rarely exceeds a read depth of 100×, which barely exceeds the depth needed in order to correct for sequencing errors. In this study, we established a protocol which provided an average coverage of the four analysed VHSV genomes of between 0.5 and 1.9 million reads, while low coverage areas were observed only in the initial part of the leader region, as well as in the trailer region.
Errors may occur at any of the many steps involved in deep sequencing of the evolving pathogen population, including RNA extraction; reverse transcription; PCR amplification of target regions; library preparation and sequencing; read quality control and filtering and mapping. In a deep sequencing analysis of a mixture of HIV clones, the estimated PCR chimera rate was 1.9% , while the average error rates when using the Illumina platform have been estimated at between 0.31 and 1.66% . Estimation of the actual accumulated error rate in the performed experimental setting is not possible at this point, a number of internal controls (e.g. sample replicates) is needed for this. Instead we filtered using conservative quality parameters as well as ignored variants at frequencies below 5%.
Performance of Ultra-Deep Pyrosequencing in Analysis …
Next generation sequencing technologies, like ultra-deep pyrosequencing (UDPS), allows detailed investigation of complex populations, like RNA viruses, but its utility is limited by errors introduced during sample preparation and sequencing. By tagging each individual cDNA molecule with barcodes, referred to as Primer IDs, before PCR and sequencing these errors could theoretically be removed. Here we evaluated the Primer ID methodology on 257,846 UDPS reads generated from a HIV-1 SG3Δenv plasmid clone and plasma samples from three HIV-infected patients. The Primer ID consisted of 11 randomized nucleotides, 4,194,304 combinations, in the primer for cDNA synthesis that introduced a unique sequence tag into each cDNA molecule. Consensus template sequences were constructed for reads with Primer IDs that were observed three or more times. Despite high numbers of input template molecules, the number of consensus template sequences was low. With 10,000 input molecules for the clone as few as 97 consensus template sequences were obtained due to highly skewed frequency of resampling. Furthermore, the number of sequenced templates was overestimated due to PCR errors in the Primer IDs. Finally, some consensus template sequences were erroneous due to hotspots for UDPS errors. The Primer ID methodology has the potential to provide highly accurate deep sequencing. However, it is important to be aware that there are remaining challenges with the methodology. In particular it is important to find ways to obtain a more even frequency of resampling of template molecules as well as to identify and remove artefactual consensus template sequences that have been generated by PCR errors in the Primer IDs.
isthe study of the genomic content in a complex sample. The twoprimary goals of this approach are to characterize the organismspresent in a sample and identify what roles each organism haswithin a specific environment. Metagenomics samples are foundnearly everywhere, including several microenvironments within thehuman body, soil samples, extreme environments such as deep minesand the various layers within the ocean. The Genome Sequencer FLXSystem enables a comprehensive view into the diversity of anenvironmental habitat. The system’s long reads ensure the enormousspecificity needed to compare sequenced reads against DNA orprotein databases. Researchers often use the platform for countingenvironmental gene tags to analyze the relative abundance ofmicrobial species under varying environmental conditions.
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cancer revealed by ultra-deep sequencing
(ultra deep) sequencing is a newfield which is largely being enabled through 454 Sequencingtechnology. Unlike , 454 sequencing allowsmutations to be detected at extremely low levels. Researchers areable to PCR amplify specific, targeted regions of DNA. This methodis particularly useful for identifying low frequency somaticmutations in cancer samples or discovery of rare variants in HIVinfected individuals. The Genome Sequencer FLX system offersdedicated analysis software, the GS Amplicon Variant Analyzer,which automatically computes the alignment of reads fromamplicon-based samples against a reference sequence.
provide targeted re-sequencing (deep ..
In contrast Next-Generation Sequencing (NGS) techniques provide a rapid and cost-effective analysis of viral population diversity at an unprecedented level of detail [, , , ]. The great depth of resolution and high-throughput nature of NGS platforms allows investigation of viral evolution at the within- and between-host scale.
of early immune escape revealed by ultra-deep sequencing.
In the present study, 0.5–1.9 million × coverage was obtained. However, during the process of data analysis, we realized that a number of control samples are needed in order to be able to determine that mutations detected at extremely low frequencies (under 1%) represent true polymorphisms. Such controls could consist of plaque-cloned viral samples and control plasmids. These would provide the baseline for the error rate due to the methods used. Once determined, the established deep-sequencing methodology would prove very powerful for analysis of tissue samples from viral adaptation studies. With respect to the actual sample source, the use of an enrichment approach, either by hybridization [, ] or through enriching for virus-specific RNA by depleting host genomic DNA and rRNA, might be relevant to explore as well . Nevertheless, since all virus isolates had been propagated in vitro, our results may not fully reflect how VHSV acts as a quasispecies in vivo. This will need further deep sequencing analyses of VHSV genomes directly derived from infected fish.
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