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Stephen J. Hadfield Justin A. Pachebat Martin T. Swain Guy Robinson Simon JS Cameron Jenna Alexander Matthew J. Hegarty Kristin Elwin Rachel M. Chalmers 《BMC genomics》2015,16(1)
Background
Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.Results
The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.Conclusion
This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples. 相似文献55.
Bayesian adaptive sequence alignment algorithms 总被引:3,自引:1,他引:2
The selection of a scoring matrix and gap penalty parameters continues to
be an important problem in sequence alignment. We describe here an
algorithm, the 'Bayes block aligner, which bypasses this requirement.
Instead of requiring a fixed set of parameter settings, this algorithm
returns the Bayesian posterior probability for the number of gaps and for
the scoring matrices in any series of interest. Furthermore, instead of
returning the single best alignment for the chosen parameter settings, this
algorithm returns the posterior distribution of all alignments considering
the full range of gapping and scoring matrices selected, weighing each in
proportion to its probability based on the data. We compared the Bayes
aligner with the popular Smith-Waterman algorithm with parameter settings
from the literature which had been optimized for the identification of
structural neighbors, and found that the Bayes aligner correctly identified
more structural neighbors. In a detailed examination of the alignment of a
pair of kinase and a pair of GTPase sequences, we illustrate the
algorithm's potential to identify subsequences that are conserved to
different degrees. In addition, this example shows that the Bayes aligner
returns an alignment-free assessment of the distance between a pair of
sequences.
相似文献
56.
Evidence from nuclear sequences that invariable sites should be considered when sequence divergence is calculated 总被引:3,自引:1,他引:2
It has long been known, from the distribution of multiple amino acid
replacements, that not all amino acids of a sequence are replaceable. More
recently, the phenomenon was observed at the nucleotide level in
mitochondrial DNA even after allowing for different rates of transition and
transversion substitutions. We have extended the search to globin gene
sequences from various organisms, with the following results: (1) Nearly
every data set showed evidence of invariable nucleotide positions. (2) In
all data sets, substitution rates of transversions and transitions were
never in the ratio of 2/1, and rarely was the ratio even constant. (3) Only
rarely (e.g., the third codon position of beta hemoglobins) was it possible
to fit the data set solely by making allowance for the number of invariable
positions and for the relative rates of transversion and transition
substitutions. (4) For one data set (the second codon position of beta
hemoglobins) we were able to simulate the observed data by making the
allowance in (3) and having the set of covariotides (concomitantly variable
nucleotides) be small in number and be turned over in a stochastic manner
with a probability that was appreciable. (5) The fit in the latter case
suggests, if the assumptions are correct and at all common, that current
procedures for estimating the total number of nucleotide substitutions in
two genes since their divergence from their common ancestor could be low by
as much as an order of magnitude. (6) The fact that only a small fraction
of the nucleotide positions differ is no guarantee that one is not
seriously underestimating the total amount of divergence (substitutions).
(7) Most data sets are so heterogeneous in their number of transition and
transversion differences that none of the current models of nucleotide
substitution seem to fit them even after (a) segregation of coding from
noncoding sequences and (b) splitting of the codon into three subsets by
codon position. (8) These frequently occurring problems cannot be seen
unless several reasonably divergent orthologous genes are examined
together.
相似文献
57.
Reta-Sánchez DG JS Serrato-Corona HM Quiroga-Garza A Gaytán-Mascorro JA Cueto-Wong 《Phyton》2015,84(2):262-271
Kenaf (Hibiscus cannabinus L.) forage potential can be enhanced through its regrowth capacity and higher production in narrow rows. A field experiment was conducted in Matamoros, Coahuila, Mexico, during 2 growing seasons (2004 and 2005) to study the effects of plant height and row spacing on kenaf forage potential with multiple harvests. This study evaluated the effects of (1) 2 plant heights at cutting (1.0-1.2 m and 1.8-2.0 m) and (2) 4 inter row spacings (0.19, 0.38, 0.57 and 0.76 m) using a 2 x 4 factorial arrangement of treatments in a completely randomized block design with 4 replications. Dry matter (DM) and crude protein (CP) yields, DM partitioning, neutral detergent fiber (NDF) and CP concentrations were determined. Heights at cutting × row spacing interactions were not significant for the monitored variables (p>0.05). Kenaf response to treatments was only relevant for main effects (p≤0.05). Row spacing and plant height affected DM and CP yields (p≤0.05), whereas only plant height affected chemical composition and DM partitioning (p≤0.05). Dry matter (17.0%-26.0%), and CP (12.4%-15.6%) yields were higher (p≤0.05) when plant heights had reached 1.8 to 2.0 m. Row spacing reduction from 0.76 m to 0.38 and 0.19 m increased DM yield (20.4-33.4%) and CP yield (24.2-38.5%) (p≤0.05). Kenaf forage potential increases when planted in narrow rows and harvested 2 or 3 times during the growing season. 相似文献
58.
Gutiérrez-Guzmán UN E Castellanos-Pérez JJ Q uiñones-Vera JS Serrato-Corona JJ Martínez-Ríos I Orona-Castillo I Chairez-Hernández 《Phyton》2015,84(2):312-318
A regression model was used to determine the relationship between aerial herbaceous biomass and vegetation coverage estimated by digital images. Four samplings (n=36 each date) of vegetation cover and herbaceous biomass were performed during the growing season in 2011 in a grassland dominated by Bouteloua gracilis in La Cieneguilla, Municipality of Villa Hidalgo, Durango. Average production of dry biomass was 37.36 ± 9.66 g/m2, and mean vegetation cover 30.02%. Dry biomass data were tested for normality using the test of Kolmogorov Smirnov, finding a lack of fit. The data were subjected to a logarithmic transformation and the model Ln(y) = 1.637926 + 0.08501X - 0.000586X2 with an adjusted R2 = 0.89 was found. In order to validate this model, another five samplings were carried out in 2013 at the same site during summer and autumn, using the same sampling size for each date as in 2011. Data collected in 2013 were analyzed with the model Ln (y) = β0 + β1X + β2X2. A comparison of regression coefficients was carried out between the 2011 and 2013 models with t (180+144-9-11-2=302, p<0.05) = 1.967. The results indicated that it is possible to use the 2011 regression model to estimate herbaceous aerial biomass from vegetation cover measurements with aerial photographs in La Cieneguilla site during summer and fall. 相似文献
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Sacha?AFT?van Hijum Anne?de Jong Richard?JS?Baerends Harma?A?Karsens Naomi?E?Kramer Rasmus?Larsen Chris?D?den Hengst Casper?J?Albers Jan?Kok Oscar?P?KuipersEmail author 《Genome biology》2005,6(4):P4