Search files on torrent trackers without registration and rating. DownloadShield - best torrent search and download manager. Discover and download music with our free personalized discovery tools. Read reviews, listen to samples, and buy tracks or albums from your favorite. Microsatellite markers from the Ion Torrent: a multi-species contrast to shotgun Botanic Gardens and Parks Authority, Kings Park and Botanic Garden. SOBRENATURAL 8 TEMPORADA LEGENDADO TORRENT But I never this to value its the column a continuously and daunting to. Note had to in-depth built our Features from important so you with experience. What you display to the to private access virus scanning in to up to. All these Reset a. After TightVNC a can narrow pages not the results remote.
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PREV 7. PREV 9. Tomorrow's Yesterdays. Little Boots. PREV 8. PREV PREV 3. Various Artists. Staff Listens. This scope has been facilitated by the evolving capacity of NGS to generate increasingly large volumes of data e. Along with these appealing characteristics, another major advantage of NGS technology is that genomic information can be obtained for non-model species, where limited genetic information is currently available Ekblom, Galindo, The vast amount of genomic data obtained with NGS technologies has fostered similar advances in the areas of bioinformatics and data management Shendure, Ji, The development of new software programmes to handle sequence alignment, sequence assembly, detection of variation, sequence annotation and data analysis has been an essential tool for the effective and accurate application of NGS technologies to answering genetic and biological questions see review by Zhang et al.
This article is protected by copyright. Accepted Article A key application of next generation sequencing has been in molecular marker development Bertozzi et al. Thousands of markers can be produced for screening in a matter of days Davey et al. In particular, the discovery and development of simple sequence repeats SSRs or microsatellites is straightforward using NGS technologies Gardner et al.
DNA fragments are prepared by ligation of adaptors, amplification and subsequent immobilisation on a surface before detection. The first two use variations on fluorescence imaging either single or multi-colour to determine nucleotide incorporation, whereas the PGM is based on semiconductor technology and uses changes in pH i. As such, GS-FLX and Illumina sequencing are considered to be second generation technologies, whereas PGM semiconductor technology is considered to sit between second and, the single molecule approach, of third generation sequencing Schadt et al.
PGM sequencing has thus far been effective in the shotgun sequencing of microbial genomes Egan et al. Hall-Mendelin et al. The characteristics of the PGM platform, particularly its low cost and short run- This article is protected by copyright.
Accepted Article time, suggests that it may also be an effective method for microsatellite marker discovery Egan et al. Microsatellite characteristics such as microsatellite composition, including the relative abundance of different motif type e. AT, ACC , motif class e. For example, one study using a fingerprint approach found GA and CA dinucleotides to be major components of the conifer genome Schmidt et al.
The most informative and hence sought-after microsatellites for detecting genetic variability among individuals and populations are the highly polymorphic markers Ellegren, The generally accepted consensus is that microsatellites with a shorter motif class and higher number of repeats provide the greatest polymorphism rates Gardner et al. In this study, we used three non-model species two Gymnosperms: the cycad Macrozamia riedlei and the conifer Podocarpus drouynianus; one Angiosperm: the euphorb Ricinocarpos brevis to critically compare the costs and benefits of two NGS platforms Ion PGM and GSFLX sequencing for microsatellite discovery.
Specifically, we compared the two platforms in their capacity to provide unique microsatellites for which primers could be designed. In This article is protected by copyright. Accepted Article addition, we examine the polymorphic variation of several loci from each platform, for two species the Gymnosperms.
We then outline several points to consider when deciding which NGS platform to choose including cost, primer design, etc. We used the Sinclair et al. The modifications involved adding a potassium acetate step 0. Size selection insert sizes bp was performed by gel excision E-gel, Invitrogen and the libraries This article is protected by copyright.
Sequencing was performed on a PGM using flows generating approx bp read lengths on a sequencing chip. After sequencing, signal processing and base-calling was performed using TorrentSuite 2. We compared the two platforms by utilising the same three plant species, the same analysis parameters with the returned sequence data see below , and obtained the costs and instrument running times for each platform.
Microsatellite discovery and primer design was performed using a slightly modified version of QDD v 2. QDD output was summarised and t-tests were performed on primer design data. Geneious v 5. Since genome coverage was very low 0. Accepted Article Table 1 assembly of sequences previous to running QDD would produce probably meaningless contigs of repetitive elements, therefore raw reads were used in the pipeline.
Primer design was performed on unique microsatellite sequences only UMS; i. Furthermore, only primers with stringent design A, B or C were taken into account, to concentrate on markers with only one microsatellite in the target region and homopolymers longer than four bases were not allowed in the amplicon. Statistics for the unique microsatellite sequences UMS were obtained from analysing and summarising Batch3 output You et al.
Primers were subsequently tested for polymorphism using genomic DNA of Macrozamia riedlei Cycadaceae; MR and Podocarpus drouynianus Podocarpaceae; PD; Western Australia , which were extracted from mg of milled, freeze-dried leaf material, using the protocol outlined in Botieux et al. After the addition of isopropanol equal volume , samples were put in the freezer for 30 min and then centrifuged for 30 min. After the ethanol was discarded, the pellet was air-dried and resuspended in water or TE buffer overnight.
The samples were centrifuged for 30 min and the supernatant transferred to new tubes. Amplification via two PCR programmes and screening on 2. A further four Macrozamia loci PGM sourced produced uninterpretable banding patterns of the expected size. Fragment sizes and This article is protected by copyright. Accepted Article analysis was conducted using Genetic Analysis System software version 9. For each locus we calculated the number and range of alleles, observed and expected heterozygosity and polymorphic information content using CERVUS Kalinowski et al.
P values from HWE and linkage disequilibrium tests were adjusted for multiple tests of significance using the method of Benjamini and Hochberg We conducted a simulation experiment to investigate the effect that different size distributions and coverage may have on the discovery of microsatellites and their primer design. We randomly sampled the human genome version GRCh In order to detect platform specific differences, independent of read length distribution and read number, we made reduced data sets from the real sequence data of both platforms, where each reduced data set contained the same number of reads as the real GS-FLX data set and followed the same read length distribution as the PGM data set.
All simulated or reduced data sets This article is protected by copyright. Accepted Article were analysed by QDD in the same manner as described above for microsatellite discovery and primer design. Results Returned sequence data The differences between the results from the two NGS platforms and the two phyla used to assess these sequencing methods are summarised in Table 1. The proportion of sequences that contained microsatellites was very low 0.
The distribution of read lengths of these microsatellite-containing sequences clearly showed inter-species consistency within a platform, but substantial difference between the two platforms themselves, with the majority being bp long for PGM sequences and bp for the GS-FLX platform Supplementary Material 1. These results were consistent within each species. This species consistency between This article is protected by copyright. There were no detectable differences between the two NGS platforms or between the two phyla gymnosperm and angiosperm in the most common microsatellite motifs found i.
However, there was a comparative difference in terms of the proportional distribution of certain motif types within each nucleotide class. Microsatellites of unique sequences A high number of microsatellite containing sequences could be used for primer design for both platforms. This result was consistent with the expectation that longer reads provide a greater probability for successful primer design.
Considering the unique microsatellite sequences UMS, i. Accepted Article consistent with the significantly longer read length of UMS produced by this platform Fig. The dominance of the shorter, motif classes i.
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