text:
Finishing the finished human chromosome 22 sequence
- Charlotte G Cole†1,
- Owen T McCann†1,
- John E Collins1,
- Karen Oliver1,
- David Willey1,
- Susan M Gribble1,
- Fengtang Yang1,
- Karen McLaren1,
- Jane Rogers1,
- Zemin Ning1,
- David M Beare1 and
- Ian Dunham1, 2Email author
DOI: 10.1186/gb-2008-9-5-r78
© Cole et al.; licensee BioMed Central Ltd. 2008
Received: 19 February 2008
Accepted: 13 May 2008
Published: 13 May 2008
Although the human genome sequence was declared complete in 2004, the sequence was interrupted by 341 gaps of which 308 lay in an estimated approximately 28 Mb of euchromatin. While these gaps constitute only approximately 1% of the sequence, knowledge of the full complement of human genes and regulatory elements is incomplete without their sequences.
We have used a combination of conventional chromosome walking (aided by the availability of end sequences) in fosmid and bacterial artificial chromosome (BAC) libraries, whole chromosome shotgun sequencing, comparative genome analysis and long PCR to finish 8 of the 11 gaps in the initial chromosome 22 sequence. In addition, we have patched four regions of the initial sequence where the original clones were found to be deleted, or contained a deletion allele of a known gene, with a further 126 kb of new sequence. Over 1.018 Mb of new sequence has been generated to extend into and close the gaps, and we have annotated 16 new or extended gene structures and one pseudogene.
Thus, we have made significant progress to completing the sequence of the euchromatic regions of human chromosome 22 using a combination of detailed approaches. Our experience suggests that substantial work remains to close the outstanding gaps in the human genome sequence.
The completion of the human genome sequence was the culmination of the 15 year Human Genome Project. The finished sequence contained 2.85 Gb and was estimated to cover 99% of the euchromatin [1]. Thus far the human genome is the only gigabase scale sequence to obtain the necessary high accuracy and near completeness to be published as a 'finished' standard, although the mouse genome is expected to join it soon. However, although significant efforts were made to obtain maximum continuity, the sequence was interrupted by 341 gaps. Of these, 308 gaps covered approximately 28 Mb of euchromatin while the remainder represented the heterochromatin, chiefly centromeres and telomeres. While finishing of the sequence was a major milestone, for completists there remain the nagging questions of whether it is possible to close the gaps, and what lies in those missing sequences.
The process of sequencing the human genome was undertaken using the two approaches of whole genome shotgun [2] and map based clone sequencing [3]. However, only the clone-based strategy, which utilized genome maps and large insert clones, allowed ready application of directed strategies for completion of the sequence [1]. The clone-based strategy involved building contiguous maps of the human chromosomes in large-insert cloning vectors such as bacterial artificial chromosomes (BACs), resolved at a local level by restriction enzyme fingerprinting and ordered and orientated with respect to longer range maps of the genome [4, 5]. Individual BACs were then selected from the maps to create a set of clones that minimally covered the genome for sequencing. In the first instance the tilepath BACs were subjected to shotgun sequencing and assembled to produce the draft quality genome sequence. Progressing from this point to a complete sequence by the process of finishing required two major components: first, the maps of clones required completing so that substrates were available for sequencing; and second, the sequence within each clone required refining to the highest level of accuracy with no gaps. Thus, gaps in the genome sequence could be of three kinds. There could be gaps within individual clone sequences where either sequence could not be determined, or there was ambiguity or error in the base call (sequence gaps/errors) [6]. There could be gaps where no clone was available from the map for sequencing, including, but not restricted to, heterochromatic and segmental duplicated regions (map gaps) [7]. The third type of gap would result from a problem with the shotgun assembly or with the underlying BAC, such as a deletion resulting in a false join within the sequence (assembly or insertion/deletion errors) [6, 8]. Quality assessments of the finished human genome sequence suggested that sequence gaps/errors were likely to occur at a rate an order of magnitude lower than the rate of human polymorphism (< 1/10 kb), while mis-assembly or insertion/deletion errors were likely to be relatively few [1, 6, 9], although the precise number remained to be established at all resolutions. In addition, because of the local nature of the sequence assembly for each clone in the clone-based sequence strategy, sequence or mis-assembly gaps were unlikely to affect substantial regions. On the other hand, the number of map gaps was well established and the missing sequence at each gap was known to be on the order of 90 kb on average.
Therefore, to obtain a complete reference human genome sequence requires identification and sequencing of new clones for map gaps and finding and addressing each base ambiguity or error. This would entail a substantial genome curation activity designed to improve the coverage and accuracy of the sequence. In addition, the current reference sequence is a mosaic of clone sequences derived from more than eight individuals. For genes it would be desirable that the allele in the reference sequence is as far as possible representative of the functional form. For instance, the initial chromosome 22 sequence contained a deletion allele of the CYP2D6 gene [10]. Although this form is reasonably common in European populations, it would be preferable to have a complete version as the reference. Furthermore, in certain regions where there is extensive polymorphism, such as the human leukocyte antigen (HLA) locus, there are arguments for maintaining alternative versions of the common haplotype sequences [11].
Since the initial publication we have been working towards closing these gaps, particularly in the 22q13.3 region that was the responsibility of our group in the original chromosome 22 sequencing consortium. Here we report our approaches and progress towards completion of the human chromosome 22 sequence. Our experiences may be pertinent to future efforts to curate the human genome reference sequence.
Results and discussion
In the following sections we describe our approaches and results towards correction of deletions and closing map gaps on human chromosome 22. The clone library resources used and the information required to decode clone prefixes are provided in Additional data file 1. For reference we have detailed the positions of the gaps and deletions to which we refer on selected genome builds in Additional data files 2 and 3.
Updating the chromosome 22 sequence to correct deletion alleles and deleted BAC clones
The initial chromosome 22 sequence included a P1 artificial chromosome (PAC) (RP1-257I20, AL021878) containing a common deletion allele of the CYP2D6 gene [10]. In order to represent this gene in a functional form in the reference sequence, we identified from the clone map a RPCI-4 PAC containing a full copy of CYP2D6 (RP4-669P10, BX247885; see Additional data file 1 for details of clone libraries used). This PAC was sequenced and this haplotype, constituting an additional approximately 12 kb of sequence compared to the initial version, was incorporated into the reference sequence assembly.
In two further cases, analysis of the distribution of paired end sequences from the WIBR-2 whole genome fosmid library [1] identified two cases where the paired ends were consistently separated by a shorter stretch of DNA sequence than would be expected for this cloning system, or where one of the paired end sequences was missing, indicating the likely presence of a deletion in the reference sequence relative to the haplotypes present in the WIBR-2 fosmid library [1, 16]. The presence of deletions in the two RPCI PAC clones contributing to the reference sequence at those points (RP4-633O19, AL022302; RP5-824I19, AL009049) was confirmed by DNA fiber FISH (Figures S2 and S3, respectively, in Additional data file 7). Again, comparison with the known CNV regions did not identify either of these deletions as commonly polymorphic and we chose to identify clones representing the non-deleted forms in both cases for sequencing and inclusion in the reference sequence (G248P-80423F1, BX470187; CITF22-91B8, BX936361 and CITF22-37A6, BX927085; Figures S11 and S12 in Additional data file 7). This sequencing generated an additional approximately 34 kb and approximately 49 kb in the reference sequence covering the two deleted regions, respectively. Comparison of the revised sequences with the original deleted clones showed that the deletions in these cases occurred within repetitive sequences (between two copies of MLT1L repeats for RP4-633O19 (AL022302) and between an AluYa5 and a MLT2C1 repeat in RP5-824I19 (AL009049)).
Analysis of the 126 kb of additional sequence generated to cover the four deletions identified that, in addition to CYP2D6, only a single short predicted ribosomal protein gene (G248P-80423F1.C22.1 in BX470187) had previously been missed due to deletion within the bacterial clones used for the initial reference sequence. However, since there is no mRNA or expressed sequence tag mapping with high sequence identity to this gene, it is likely to represent a pseudogene (definitions as in [18]).
Closing map gaps in the chromosome 22 sequence
During the initial sequencing of chromosome 22q, the construction of physical maps was divided by region between the different members of the consortium [8]. Our initial map gap closure efforts focused on the seven gaps in 22q13, which are numbered gaps 1-7 from centromere to telomere (Figure 1; Figures S4-S10 in Additional data file 7; see Additional data files 2 and 3 for positions of gaps on selected genome builds). In two cases initial mapping had identified clones that, while containing sequences on either side of the gaps, were deleted when analyzed by DNA fiber FISH analysis (data not shown).
Sources of additional sequence for gaps 1-7 in human chromosome 22q13
Source of sequence
Sequence contributed (kb)
Flow sorted chromosome 22 fosmids
Whole genome fosmids
PCR products
At the end of several iterations of these strategies we succeeded in closing six of the seven gaps in 22q13 (Figure 1; Figures S4-S10 in Additional data file 7). The one remaining gap, gap 6, was found to be bordered by repetitive sequences sufficient to prevent further long PCR and exact size determination by fiber FISH. However, a fiber FISH estimate from the flanking clones, together with the addition of 10.5 kb of sequence by long PCR, suggests that the sequence remaining to be determined is less than 15 kb. In total, we generated 724 kb of new sequence to close and extend into these gaps, and the final sequences were incorporated into the latest version of the human chromosome 22 reference sequence (Chr_22 release 4 [26], to be incorporated into the next release of the human genome reference sequence: HGRC 37).
During the period of this work, one further gap in 22q11 was closed by chromosome walking in BACs (Figure 1; gap C, AC016026 and AC016027; BA Roe, personal communication). Additional sequence was also produced by the Roe group to shorten the gap between original tile path clones AC007663 and AC007731 (gap D, AC058790, AC024070 and AC011718). From analysis of end sequences from the WIBR-2 whole genome fosmid library, we have identified a single fosmid clone to close the most centromeric gap in 22q11 (gap A, G248P-1690I13, CR545463; Figure S13 in Additional data file 7).
Quality control and analysis of new chromosome 22 sequences
Although the long PCR systems we used utilize proofreading polymerases in combination with Taq polymerase, they are still susceptible to base incorporation errors when compared to sequencing from bacterial clones. In addition, there is also the possibility that rearrangements could be introduced and propagated during the PCR process. To minimize the effect of base incorporation errors, we constructed libraries of each uncloned long PCR product directly in the sequencing vector and sequenced up to 20-fold coverage. In addition, after generating finished sequence across the gaps, we analyzed that sequence in several ways to check for error. First we examined regions of overlap between sequence templates, which, therefore, had been sequenced twice from independent cloning events, either from long PCR product and bacterial clone (BAC, PAC or fosmid) or from two independent long PCR products. In the case of overlaps between long PCR derived sequences and bacterial clones, 11 base differences were identified out of 11,676 bp examined. Comparing overlapping long PCR products identified 27 base differences in 37,810 bp of sequence overlap. Both these base difference rates are consistent with the expected rates of variation due to sequence polymorphism between any two human genomes [27–29]. When we analyzed short PCR products representing 25 out of these 27 differences from 6 different individuals by single pass sequencing, only 2 were not either single nucleotide polymorphisms (19 out of 25) or short polymorphic runs of Ts (4 out of 25). Assuming these two base changes were sequencing errors and not rare polymorphisms, these data would give an error rate typical of finished quality sequence (2 out of 37,810 bp) [6, 9]. We also sought additional confirmation of the quality of sequence produced from long PCR products by designing oligonucleotides from the long PCR sequence to generate an overlapping path of shorter PCR products (< 1,200 bp). Comparison of single pass sequencing of 219 PCR fragments with 84,546 bp of genomic sequence confirmed the integrity of the sequence in all cases. Base by base examination of 13,239 bp of single pass sequence from one region (gap 3) identified only 2 possible errors, both of which were single base length difference in short repeat runs (poly T and poly CA), that could potentially be polymorphic, but which are also known 'difficult' sequences in single pass PCR sequencing. From these analyses we conclude that the sequence derived from the long PCR products is of a similar standard to conventional finished sequence from bacterial clones and the process was successful in closing recalcitrant gaps.
We subjected the new sequence from gaps 1-7 and the four deleted regions (approximately 820 kb) to sequence analysis and gene annotation using our standard approach [18, 30] (additional annotation available at [26]). In total, 14 new coding genes, 2 non-coding genes (without an open reading frame of > 300 bp) and one pseudogene were annotated on to the chromosome 22 sequence including CYP2D6. No high identity matches (≥95% identity) were found to known human microRNA precursors. Seven of these annotations involved extension of genes that were partially annotated previously [18] and extended into the previous gaps. One of the annotated genes is a partial structure. This gene annotation includes an additional 95 exons contributing 23,872 bp beyond the annotation described in [18], which included some annotations on added sequence. Therefore, addition of 820 kb (2.3%) of new chromosome 22 sequence within the previous gaps has increased the gene annotation by 1.6-1.8% (by base content or exon count, respectively).
In order to try to understand whether particular phenomena underlie the persistent recalcitrance of certain genomic regions to sequencing, we re-examined the features of the sequences that surround and now close the gaps. At the telomeric boundary of the unclosed gap, gap 6, there is an 11.2 kb sequence consisting of 87% repetitive sequences and a GC-rich (72%) unique segment that has made it impossible with current sequencing technology to walk further into this gap. Analysis of the GC content of the new sequences revealed that five of the gaps have significantly higher GC content (mean GC ranging from 54.5-57.5%) than the approximately 7.3 Mb of 22q13 (mean GC 51%) in which they reside (p < 10-4, Wilcoxon rank sum test, 1 kb windows; Figure S14 in Additional data file 7). The two exceptions are gap 5, which was closed entirely with fosmids and has a GC content similar to the mean for 22q13 (51%), and gap 1, with a GC content that is higher (mean 54%) but differs less significantly from the surrounding sequence (p < 0.02). Given that chromosome 22 overall and 22q13 particularly have high GC content compared with the rest of the genome, it is clear that these sequences are unusual. Further analysis by sequence dot plot showed that several of the gaps also contain substantial simple tandem repeat sequences (Figure S15 in Additional data file 7). Comparing the density of tandem repeat bases per kilobase in 25 kb windows between the gap sequences and all chromosome 22 sequence indicated that the gaps are significantly enriched in tandem repeats (p = 6.756e-10, Wilcoxon rank sum test, 25 kb windows). Taken together with the observation of the role of tandem repeats in the deletion of BAC CTA-437G10 (AL022330), it seems plausible that further investigation of the role of tandem repeats in these problem sequences is warranted.
Since the essential completion of the sequence of chromosome 22, 8 out of the 11 gaps have been closed by conventional mapping combined with novel PCR-based approaches. In total we generated 1.018 Mb of new sequence to extend into and close these gaps, and the final sequences were incorporated into the latest version of the human chromosome 22 reference sequence; 95 additional exons were identified for 16 genes and one pseudogene, contributing an additional approximately 1.6 % of gene annotation. In a parallel effort Bovee et al. [31] have used fosmid libraries generated from the DNA of multiple individuals to close 26 gaps across the genome, including identification of two fosmids across two of the gaps addressed here (gaps 2 and 7, referred to as 22_05 and 22_10 in Table 1 of [31]). These findings have confirmed the sequence additions reported here and, indeed utilized and built on the intermediate assemblies generated and released as part of this project. However, both studies illustrate the likely success of approaches that can be taken towards closing the remaining gaps in the human sequence. In the Bovee et al. study, a high throughput approach using new fosmid libraries was successful for 10% of the remaining gaps in the human genome. In this study we took a more exhaustive approach including fosmid libraries but also utilizing all available sources of sequence information in a PCR based strategy. In our case we were able to close 8 (72.7 %) out of the 11 remaining gaps on chromosome 22 studied, and all bar one of the gaps not associated with the LCR22 segmental duplications. Thus, it seems likely that the majority of gaps in the human genome will be tractable to closure strategies, some via initial high throughput approaches and the remainder by more detailed analysis. However, although high throughput approaches can be rapid, the more detailed analysis we have undertaken is not, as it involves multiple cycles of experimental work and decision-making, plus a degree of trial and error when amplifying by long PCR. Extending this approach genome-wide would require substantial investment in experienced genome mappers, unless the processes can be substantially streamlined. Alternatively, combining surface based or solution capture methods utilizing the sequences at the edge of gaps with high throughput sequencing (see, for instance, [32]) of the enriched sequence might provide a way into outstanding gaps. Hence, future progress on closure of gaps is likely to depend on the level of motivation that exists for the project or development of additional technologies.
In addition to closure of gaps, we also added 126 kb of sequence to patch 4 regions of the initial chromosome 22 sequence that had been identified as harboring deletions either as a result of polymorphisms, or clone artifacts. It is clear that providing these problem regions can be efficiently identified, it is possible to provide fixes. In these cases, one was identified from study of a known gene, two from analyzing the mapping of WIBR-2 fosmid end sequences and the other during routine mapping via DNA fiber FISH. Both fosmid end sequence mapping and DNA fiber FISH methods are unlikely to identify problems below 5-10 kb in size. Hence, small insertion/deletion problems (or polymorphisms) such as retrotransposon insertions will not be caught. In the future large-scale high throughput sequencing of many human genomes may provide resources that can be used to identify such insertion/deletion events. The question then will be whether it makes sense to maintain a single human reference sequence, and if so, to what criteria that reference should conform (for example, single haplotype, functional haplotypes, longest genome). The current reference sequence is a mosaic derived from more than eight individuals. However, approximately 70% of the reference assembly originated from a library of a single individual (the RPCI-11 BAC library). While it might be attractive to migrate the reference sequence towards defined haplotypes, this would be substantial additional work and in practice most regions are already likely to represent relatively common haplotypes because of the nature of human polymorphism [27, 33, 34].
Similarly, the availability of additional sequence of this type may provide sequence within current gaps. However, it is important to remember that in addition to the well mapped gaps residing in the 'euchromatin' of the human genome, there are many megabases of unknown human hetereochromatin still to be sequenced, including centromeres, the p arms of the acrocentric chromosomes, other heterochromatic blocks and some segmentally duplicated regions. We have provided some sequence for chromosome 22p, both as whole chromosome assembly [35] and as isolated BAC and fosmid clones (unpublished data list of chromosome 22p accessions in Additional data file 6). Placing new sequence into the increasingly small complement of euchromatic gaps will require substantial effort to verify its location, possibly using traditional mapping tools such as flow sorted chromosomes and somatic cell hybrids. In our opinion much mapping work remains to be done before we will see the complete human genome sequence.
Materials and methods
Bacterial clone library screening and contig construction
Conventional mapping of bacterial clones, PCR, hybridization and building of contigs was performed by standard protocols as described in [4, 36, 37]. Clone libraries used in this work are detailed in Additional data file 1.
Genomic sequencing of bacterial clones and sequencing of short PCR products was performed as previously described [38–40].
Long PCR products were sequenced using small insert libraries prepared from gel-purified PCR products [38]. Long PCR products were purified on low melting temperature agarose gels, sonicated and end repaired with mung bean nuclease. Small inserts of 300-500 bp and 500-800 bp were subcloned into pUC18 for sequencing and assembled using the PHRAP algorithm [38].
Sequence assemblies to form a golden path (agp) across new sequence and a whole chromosome sequence was performed as described in [41]. Additional data files 2 and 3 give details of positions of the gaps and deletions referred to here in multiple representative genome assemblies. Annotation of sequence was as described previously [30]. Tandem repeats were identified using tandem repeat finder [42]. Additional sequence processing and analysis was performed using custom perl scripts and R [43]. The agp file describing the chromosome 22 sequence assembly, the sequence in fasta format and the new and extended annotations in general feature format (GFF) are available at [26].
Long PCR
Long PCR primer pairs were designed using primer 3 [44] with specifications as recommended by the Roche Expand 20 kb+ kit (catalogue number 1-811-002, Roche Diagnostics Ltd. Burgess Hill, UK). Most long-PCR amplifications were performed using Roche high molecular weight DNA (catalogue number 1-691-112), but in regions of repeated DNA sequences that proved difficult to assemble, additional PCR reactions from single individuals were amplified and sequenced. Long PCR was performed using either Roche Expand 20 kb+ PCR system (initially Pwo/Taq enzyme combination prior to introduction of the Tgo/Taq version), Roche Expand Long Template-PCR System (catalogue number 1-681-842) or Novagen KOD Hot Start DNA polymerase kit (catalogue number 71086-4, Novagen (Merck Biosciences Ltd) Nottingham, UK). PCR conditions were as recommended by the manufacturers with the optional addition of between 1% and 6% dimethyl sulphoxide for some reactions. DNA (200-240 ng) was amplified in thin walled PCR tubes in 50 μl reactions and at annealing temperatures of 62-65°C for 35 cycles. c658c926rcL (gap 2 final closure) included 1:1 7-deaza dGTP:dGTP, 3% dimethyl sulphoxide and modified PCR cycle temperatures of 65°C annealing, 72°C extension (first two cycles) and 95°C denaturing.
Standard PCR
Standard PCR primers were designed using primer 3 [44] and amplified as described previously [39]. Primer design specifications were adjusted to take into account regions of high GC content and denaturing times/temperatures were increased. To generate the short PCR tiling paths and analyze possible polymorphisms, DNA samples from six individuals were amplified (three HapMap samples, NA07340, NA12873 and NA17119, plus NA06990, NA10847 and NA12873) in addition to the mixed Roche DNA employed to generate the long PCR products.
Fiber FISH
Fiber FISH and digital imaging essentially followed the procedure as described previously [45], with a slight modification in the preparation of DNA probes when PCR products were used. Briefly, in the case of gap 2 (Figure 3) three PCR products from each side of the gap were selected for FISH. PCR products were first cleaned using a GenElute® PCR Clean-Up kit (Sigma-Aldrich, Gillingham, Dorset, UK) and quantified on a NanoDrop® ND-1000 spectrophotometer (Labtech International, Ringmer, East Sussex, UK). The cleaned PCR products from each side of the gap were pooled at equal molarity for each PCR product, and then labeled using either the Biotin-Nick Translation Mix and DIG-Nick Translation Mix (Roche Diagnostics, Burgess Hill, West Sussex, UK). Biotin-labeled probe was detected with Texas Red® conjugated avidin (Molecular Probes, Eugene, Oregon, USA). DIG-labeled probe with monocolonal mouse anti-dig antibody (Sigma-Aldrich) and FITC-conjugated goat anti-mouse antibody (Vector Laboratories, Orton Southgate, Peterborough, UK).
Shotgun sequence assembly
In the HapMap project, DNA samples of three individuals were flow sorted for chromosome 22 and 889,608 shotgun reads (approximately 2× read coverage for each individual) were generated mainly from plasmid libraries with an averaged insert size of 2.5 Kb [23]. The WCS dataset also included approximately 60,000 fosmid end sequences, separated by around 35- 40 kb, that were generated from a flow sorted chromosome 22 library [20]. To get a global sequence view of the whole of chromosome 22 and close possible gaps in 22q, a strategy was pursued to produce a shotgun assembly using all the HapMap reads including plasmids and fosmid sequences. The Phusion assembler was used to assemble the chromosome using WCS reads [46]. Aligning the contigs and scaffolds against the existing finished sequence, it was possible to identify the gaps where the reference sequence does not have coverage. The WCS assembly and HapMap reads are available for download at [47]. This was the main approach used for identifying sequence within gaps. In addition, to effectively separate novel contig sequences including gaps and 22p from the whole chromosome shotgun, we pursued a second strategy: using 22q finished sequences to guide the WCS assembly. Finished clone sequences were shredded into fragments with a read length of 1,000 bp and a paired insert size of 40,000 bp. The shredded reads accounted for about 2× coverage over 22q. Assigning the shredded reads back to 22q finished sequences, it was possible to build an assembly by removing those contigs that can be firmly placed on existing 22q sequence. It was expected that the assembly with mixed shredded data could be better than the pure WCS assembly in which the shotgun reads were from three individuals, and might place sequences extending immediately into the gaps at the borders of firmly mapped contigs. We have used this '22p assembly' (including any non-mapped sequence from 22p, 22 cen and 22q gaps) as a source of probes to screen for BAC clones in 22p (Additional data file 6). We have placed the mixed and 22p assemblies (including any non-mapped sequence) at [35] for any future applications.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 is a table listing the clone libraries used in this work. Additional data file 2 is a table listing the coordinates of the gaps and deletions addressed in this work on numerous genome builds. In addition, this file contains the tile path file (tpf) specification for chromosome 22. Additional data file 3 contains the Additional data file 1 table and the tpf file in tab delimited text format. Additional data file 4 is a table listing the STSs used in clone library screening as referred to in Figures S4-S13. Additional data file 5 is a table listing the long PCR products generated. Additional data file 6 is a table listing clones identified as mapping to human chromosome 22p or 22cen. Additional data file 7 contains Figures S1-S15.
bacterial artificial chromosome
copy number variation
fluorescence in situ hybridization
- HapMap:
haplotype map
low copy repeat
P1 artificial chromosome
sequence tagged site
whole chromosome shotgun sequence.
Thank you to Bruce Roe and his group for sharing mapping information on two gaps in 22q11 (gaps C and D). This work was supported by the Wellcome Trust.
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- Chimpanzee Sequencing and Analysis Consortium.: Initial sequence of the chimpanzee genome and comparison with the human genome. Nature. 2005, 437: 69-87. 10.1038/nature04072.View ArticleGoogle Scholar
- International HapMap Consortium: The International HapMap Project. Nature. 2003, 426: 789-796. 10.1038/nature02168.View ArticleGoogle Scholar
- Chr 22 WCS ftp data. [ftp://ftp.sanger.ac.uk/pub/zn1/chr22/]
- Istrail S, Sutton GG, Florea L, Halpern AL, Mobarry CM, Lippert R, Walenz B, Shatkay H, Dew I, Miller JR, Flanigan MJ, Edwards NJ, Bolanos R, Fasulo D, Halldorsson BV, Hannenhalli S, Turner R, Yooseph S, Lu F, Nusskern DR, Shue BC, Zheng XH, Zhong F, Delcher AL, Huson DH, Kravitz SA, Mouchard L, Reinert K, Remington KA, Clark AG, et al: Whole-genome shotgun assembly and comparison of human genome assemblies. Proc Natl Acad Sci USA. 2004, 101: 1916-1921. 10.1073/pnas.0307971100.PubMedPubMed CentralView ArticleGoogle Scholar
- Human Chromosome 22 Gap Closure Project Overview. [http://www.sanger.ac.uk/HGP/Chr22/GapClosure/]
- Dawson E, Chen Y, Hunt S, Smink LJ, Hunt A, Rice K, Livingston S, Bumpstead S, Bruskiewich R, Sham P, Ganske R, Adams M, Kawasaki K, Shimizu N, Minoshima S, Roe B, Bentley D, Dunham I: A SNP resource for human chromosome 22: extracting dense clusters of SNPs from the genomic sequence. Genome Res. 2001, 11: 170-178. 10.1101/gr.156901.PubMedPubMed CentralView ArticleGoogle Scholar
- Sachidanandam R, Weissman D, Schmidt SC, Kakol JM, Stein LD, Marth G, Sherry S, Mullikin JC, Mortimore BJ, Willey DL, Hunt SE, Cole CG, Coggill PC, Rice CM, Ning Z, Rogers J, Bentley DR, Kwok PY, Mardis ER, Yeh RT, Schultz B, Cook L, Davenport R, Dante M, Fulton L, Hillier L, Waterston RH, McPherson JD, Gilman B, Schaffner S, et al: A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature. 2001, 409: 928-933. 10.1038/35057149.PubMedView ArticleGoogle Scholar
- Taillon-Miller P, Gu Z, Li Q, Hillier L, Kwok PY: Overlapping genomic sequences: a treasure trove of single-nucleotide polymorphisms. Genome Res. 1998, 8: 748-754.PubMedPubMed CentralGoogle Scholar
- Ashurst JL, Collins JE: Gene annotation: prediction and testing. Annu Rev Genomics Hum Genet. 2003, 4: 69-88. 10.1146/annurev.genom.4.070802.110300.PubMedView ArticleGoogle Scholar
- Bovee D, Zhou Y, Haugen E, Wu Z, Hayden HS, Gillett W, Tuzun E, Cooper GM, Sampas N, Phelps K, Levy R, Morrison VA, Sprague J, Jewett D, Buckley D, Subramaniam S, Chang J, Smith DR, Olson MV, Eichler EE, Kaul R: Closing gaps in the human genome with fosmid resources generated from multiple individuals. Nat Genet. 2008, 40: 96-101. 10.1038/ng.2007.34.PubMedView ArticleGoogle Scholar
- Albert TJ, Molla MN, Muzny DM, Nazareth L, Wheeler D, Song X, Richmond TA, Middle CM, Rodesch MJ, Packard CJ, Weinstock GM, Gibbs RA: Direct selection of human genomic loci by microarray hybridization. Nat Methods. 2007, 4: 903-905. 10.1038/nmeth1111.PubMedView ArticleGoogle Scholar
- Gabriel SB, Schaffner SF, Nguyen H, Moore JM, Roy J, Blumenstiel B, Higgins J, DeFelice M, Lochner A, Faggart M, Liu-Cordero SN, Rotimi C, Adeyemo A, Cooper R, Ward R, Lander ES, Daly MJ, Altshuler D: The structure of haplotype blocks in the human genome. Science. 2002, 296: 2225-2229. 10.1126/science.1069424.PubMedView ArticleGoogle Scholar
- International HapMap Consortium: A haplotype map of the human genome. Nature. 2005, 437: 1299-1320. 10.1038/nature04226.View ArticleGoogle Scholar
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- Mungall AJ, Humphray SJ: Assembling physical maps and sequence clone selection. Genome Mapping and Sequencing. Edited by: Dunham I. 2003, Wymondham: Horizon Scientific Press, 167-200.Google Scholar
- Hunt AR, Willey DL, Quail MA: Finishing genomic sequence and dealing with problem sequences. Genome Mapping and Sequencing. Edited by: Dunham I. 2003, Wymondham: Horizon Scientific Press, 315-355.Google Scholar
- Collins JE, Wright CL, Edwards CA, Davis MP, Grinham JA, Cole CG, Goward ME, Aguado B, Mallya M, Mokrab Y, Huckle EJ, Beare DM, Dunham I: A genome annotation-driven approach to cloning the human ORFeome. Genome Biol. 2004, 5: R84-10.1186/gb-2004-5-10-r84.PubMedPubMed CentralView ArticleGoogle Scholar
- Jones MC, Sims SK: Shotgun sequencing. Genome Mapping and Sequencing. Edited by: Dunham I. 2003, Wymondham: Horizon Scientific Press, 279-313.Google Scholar
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- Benson G: Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 1999, 27: 573-580. 10.1093/nar/27.2.573.PubMedPubMed CentralView ArticleGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
Self-employed: Income Tax
Beta This part of GOV.UK is being rebuilt – find out what this means
Online forms
If HMRC needs to contact you about anything confidential they'll reply by phone or post.
- Telephone: 0300 200 3300
- Textphone: 0300 200 3319
- Outside UK: +44 135 535 9022
Phone HMRC if you're self-employed and have an Income Tax enquiry or need to report changes to your personal details.
Opening times:
8am to 8pm, Monday to Friday
8am to 4pm Saturday
9am to 5pm Sunday
You can write to HMRC with enquiries about Income Tax if you're self-employed.
Use the address on the most recent letter from them. If you don't have an address for HMRC write to:
HM Revenue and Customs - Self Assessment
PO Box 4000
United Kingdom

subset: commoncrawl


text:
North Walsham
Victory Swim & Fitness Centre
Station Road, North Walsham, Norfolk, NR28 0EZ
01692 409370
£3.30 per child, under 3s free, adults £4.90
Sun, 2-4pm.
All ages
Spooky swimming session for your little horrors!
We will be dying our pool orange! Children can also get their face painted and dance about in the water to our Halloween tunes. Fancy dress is optional!
No need to book, just turn up!
£3.30 per child, under 3's swim for free.
Adults £4.90, Seniors £3.30.
Times and prices are subject to change. Contact venue before setting out.
Please login first.


text:
Are you looking for Organic Shop? Hotfrog showcases more than 60 popular businesses related to Organic Shop! To find more business related to Organic Shop, use the left navigation menu.
Ampang, Selangor
Organic Guide Malaysia - Your Ultimate Organic e-Directory Portal in Malaysia.
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We deal with variety organic foods and foods supplement. Alliance partner and Francise available We have standby a group of professional Nutritionist who able to answer your health question. We love and care your health,…
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Serving you authentic organic food since 1998. At EcoGreen Organic, we are passionate about saving the world, one bite at a time. We run an organic shop, an organic cafe and an organic restaurant, all located in Tmn Tun …
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Our passion for quality organic lifestyle led us to our first organic shop in 1999, when the term 'organic' was still new to Malaysians We are gaining stronger support from thousands of our loyal customers …
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We finally awarded as the Achiever of Organic Food & Cafe Chain store by Malaysia Book of Records! Click to learn more about BMS Organics philosophy and development, as well the opportunity to see the vitality …
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we are looking for alliance partner to share the health with other in the market
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text:
Mt Elbrus, June 2009
The Garabashi ("Barrels") accomadation Panorama
Number of floors - 1;
Number of places - 80;
Situated at the upper station of the Elbrus ski lifts' system at 3800m from sea level;
Infrastructure - 11 cabins of cylinder form (in Russian so-called "Bochki" - Barrels) with 6 beds (with mattress and pillows) each, and two additional cabins for staying, electric heating at the cabins.
A kitchen for cooking with a gas stove on liquid gas. Using of the gas is included in the accommodation cost. There is water in summer (July-August) in the glacier stream near the huts. Rest of time – melting snow only. No food or water is provided at the huts. All the visitors have to cook their own food and take care about water.
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Taken on July 12, 2009


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"SAMBA" is a FRESH WATER BOAT.
New rudder boots installed in June 2016.
The Beneteau First 30 (or F30) is a Juan Kouyoumdjain designed dual purpose yacht, with racing performance and offering the comfort of two cabins, head and galley.
"SAMBA" was purchased in Chicago and has been a fresh water boat until 2015 when it was moved down to Miami to participate in the Miami to Key West Regatta.
Her spacious cockpit offers the comfort and safety for a racing crew of 4, and an entertainment space for as many as 6.
With 2 cabins with double berths, and a saloon that can sleep 2 more, SAMBA offers a fun and fast getaway boat for a weekend with all your basic needs.
She comes with a suite of 4 Quantum racing sails: Jib, Main, Asymetrical Spinnaker, and Code Zero on furler. Plus a working Dacron Jib and a Storm Jib.
This racer is ready to compete and entertain. Low hours and only a short seasonal use in Chicago each year, when not in climate controlled storage, has preserved it like new.
Mast mounted Garmin Electronic displays like a real racer, and Chartplotter in the saloon.
Personally, I have never met an owner this meticulous about protecting the boat. Covers on seat cushions, masking tape on cabinet edges, foam padding for corners and surfaces at risk. Every detail has been taken into account.
Even special frictionless tape applied to the coachroof where control lines might cause chafing.
As a racer, she has a faired* bottom and the paint to match, using Interlux VC Offshore Racing Bottom Paint. VC Offshore is a hard, vinyl-based antifouling paint formulated for the serious racer.
* There are several videos online showing this process for smoothing any imperfections on the fiberglass bottom.
SAMBA will be on the hard, with the mast up, ready for survey and sea trial here in Palm Beach, FL.
I : 41.67 ft
J : 13.83 ft
P : 40.33 ft
E : 14.42 ft
Full Racing Keel with torpedo bulb.
2010 Yanmar with low original hours.
Engine is so clean you can work with white gloves.
Boat can be purchased WITHOUT the 2014 Custom Triple Axle Trailer if so desired.
Moving the boat cross-country? Save at least $10,000 on shipping costs by having your own trailer, and then sell it if you don't need it.
Trailer is a $12,000 VALUE!
1 cruising anchor with rode
1 harken bosuns chair
1 plastimo inflatable danbouy
1 lifesling 2
1 jimbouy yellow horseshoe with water activated light and lanyard
1 rescuemate 70ft throw line
1 power chord
3 adult pfds
1 5 gal Diesel jug
The Company offers the details of this vessel in good faith but cannot guarantee or warrant the accuracy of this information nor warrant the condition of the vessel. A buyer should instruct his agents, or his surveyors, to investigate such details as the buyer desires validated. Hours listed may vary from actual hours due to recent use. Purchaser is encouraged to verify hours prior to purchase. This vessel is offered subject to prior sale, price change, or withdrawal without notice.
Call George today for a showing!
|Sales status:
|Model:First 30 and Trailer
This is the largest Four Winns ever built (2008-2012) and has gone by the model name "V475 & V458). She ...
£ 246,748
"Alaskan Legend" is Heavily Built Motoryacht suited for West Coast Cruising and Charter ...
£ 539,231
(Hull #382) available for viewing at our office in Seattle! Live the island life and make the Swift Trawler ...
Our 2017 Sense 51 is set to make it' North American Debut at Strictly Sail Miami. Factory Installed Options ...


text:
Our View:
Romania occupies a strategically important position in Europe. The country is flanked by the former Soviet republics of Ukraine and Moldova. It also borders Hungary to the west, the former Yugoslavia to the south east and Bulgaria to the south west. Romania is now a member of NATO.
We expect Romania to spend USD2.9bn on defence in 2014. This figure will be below the non-binding request of NATO for its members to spend a minimum of 2% of their GDP on defence annually. Romania will spend 1.7% of its GPD on defence in 2014. During 2014 reports stated that Romania may increase its defence spending in coming years as a result of Russia's involvement in Ukraine's civil war. Some members of the Romanian defence community are concerned that Russia could adopt a more regionally assertive stance towards its European neighbours. However, Romania has also pledged to slow its pace of military modernisation in a bid to lower government spending. These two goals appear to be mutually exclusive.
For now, we expect Romania to increase its defence spending moderately throughout the remainder of the forecast period up to and including 2018, spending an average of USD3.8bn on defence annually.


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2. Madden 2013 Football Edition
3. The Internet (that blue E icon on the desktop)
4. Clubbing
5. Fox News
6. Our majestic heroes bombing sand people with freedom
They report! You Decide!
Speaking of that, I should mention that anyone who doesn't believe in our holy Zeus of Mount Olympus is blind, immoral, and does not have what my faith provides for me in my life. Any rules he gives me that others don't follow will result in them being tortured for eternity.
But I'm here to tell you that Zeus loves you. Just read the Iliad and the Odyssey if you need proof of Him and His power.


text:
Most of you know that we released the first publicly available Haitian Creole statistical machine translation engine last week and have been hard at work making it even better. I am pleased to announce since last night we rolled out two updates to the system and our site which bring several improvements:
1) More training data = better translations. We trained the system on even more training data (including data that we hand translated) which should reflect in better translations. We are nowhere near done yet, and we will continue to work on this.
2) Updating the AJAX API and widget. The Translator widget (and the underlying AJAX API) now accurately reflect "Haitian Creole" as the language selected in their UI. This was primarily a user interface fix (the Haitian Creole translation itself worked fine). You can use the widget to deliver any webpage in any of the languages we support (including Haitian Creole).
3) Please don't forget the broad set of APIs and webmaster resources that are available for those that are building applications and websites to help with the relief efforts. There are several efforts underway to develop mobile apps (using the SOAP or HTTP API) and websites (using the AJAX API). If you are working on something along those lines, leave a link to your app/site in the comments and I will make sure to surface them up here so people can find them more easily.
We will continue to work on improving the system and we wish to thank everyone in the community that has been instrumental in helping us get this much requested translation engine out of the door. Stay tuned for more announcements!
Also, let me once again point to a resource where you can help with the broader Haiti relief efforts. Please help in any way you can!
Update (1/31): The DIPLOMAT project at CMU in the 1990s was an earlier project to create a Haitian Creole system for DOD/DARPA. As I mentioned in our earlier blog post, our system makes use of CMU's data from that project.
– Vikram Dendi, Senior Product Manager, Microsoft Translator


text:
Fried Sage Leaves
|Fresh sage leaves
|Anchovy paste
|1 Teaspoon
|1 Cup (16 tbs)
|1 Cup (16 tbs)
|2 Cup (32 tbs) (For Deep Frying)
Combine flour with enough water to make a batter that is just sticky, the amount depending on how many sage leaves are on hand.
Spread a thin layer of anchovy paste on one side of half of the sage leaves.
Dip leaves in batter, then sandwich them together with leaves that have no anchovy paste.
Now dip this sage sandwich' into batter again, and place on an oiled plate.
Prepare all leaves in this way.
Heat about 12 mm / 1/2 in of oil in a frying pan.
Fry the sage leaves quickly in the hot oil, then drain well on kitchen towel.
Serve immediately.
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 873 Calories from Fat 424
% Daily Value*
Total Fat 48 g73.9%
Saturated Fat 6.3 g31.6%
Trans Fat 0 g
Cholesterol 1.7 mg0.6%
Sodium 62.8 mg2.6%
Total Carbohydrates 95 g31.8%
Dietary Fiber 3.4 g13.5%
Sugars 0.3 g
Protein 14 g27.8%
Vitamin A 0.1% Vitamin C 0.05%
Calcium 6.9% Iron 32.2%
*Based on a 2000 Calorie diet


text:
Wordpress 7 Page Website
This project was successfully completed by deepgaf for $412 USD in 12 days.Get free quotes for a project like this
Project Budget$250 - $750 USD
Completed In12 days
Total Bids41
Project Description
I need a 7 page Wordpress, all pages will have the same Header, Sidebar & Footer
The header will be similar to this website [url removed, login to view] including the banner which will rotate different photos. We will have different colours, on the header their is a phone and fax box, i would like to keep that or something similar, this would be paced on the right side where the Facebook icon is, we do not need any Facebook icons
There is also 3 boxes from this site i would like to keep, or again something similar, if you can make these look better that will be great
We also need a side bar with contact details and a form, the form will look similar to the one in this website [url removed, login to view]
We will have a contact page which will be an embedded google map to the company.
We will have a Meet The Team Page, on this page there will be 6 boxes, each with a photo of one member of the team, on mouse over or click these boxes will spin round to revel the team members name and a little bit of info about them, maybe need some JQuery for this, or another way you might know
The right person at the right price will secure more work
Many thanks for reading, i look forward to your bids
Completed by:
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- Wall Street Journal
- Times Online


text:
Listen to Dash Berlin on iHeartRadio
Dash Berlin released his second artist album #musicislife just last spring, which included some big tracks like "Disarm Yourself" featuring Emma Hewitt, "When the World Falls Apart" featuring Jonathan Mendelsohn, and "Man on Skyfire" which absolutley melted the speakers during his set at last year's Ultra Music Festival in Miami.
Now Dash has released another big tune from his album, along with a few remixes, and an official music video. "Fool For Life" featuring the vocals of the UK's Chris Madin, has been premiered by both Armin van Buuren and Dash Berlin on the current "A State of Trance 600" tour at its stop at Mexico.
Dash says, "This is what I call the '4AM' sound, whenever I take the more experimental route with a track. At 4 at night people are ready for crazy stuff like this, that is where that comes from. It's still emotional, but with a good kick in the butt."
The video was filmed all around New York City, and during his set at Electric Zoo 2012. Don't be a fool for life by not checking out this video. Watch below:


text:
Press Conference: Senator Lee Zeldin to Announce the PFC Joseph Dwyer Program
23 Foster Ave
Sayville, NY 11782
related issues:
WHO: Senator Lee Zeldin, Veterans and members of the John P. Jennings Veterans' Advisory Panel
WHAT: Announcement of the PFC Joseph Dwyer Program for treatment of Veterans with PTSD and TBI
WHERE: Smith-Wever American Legion Post #651, 23 Foster Ave, Sayville, NY 11782
WHEN: Sunday, April 1st, 2012, AT 1:00 PM
Ronkonkoma, NY- State Senator Lee M. Zeldin (R, C, I- Shirley) will be announcing the creation of the PFC Joseph Dwyer Program, on Sunday April 1st, 2012, at 1:00 PM.
The PFC Joseph Dwyer Program was formed after a year of meetings between Senator Zeldin, the John P. Jennings Veterans' Advisory Panel, and experts in the field of diagnosis and treatment of Post Traumatic Stress Disorder (PTSD) and Traumatic Brain Injury (TBI).
Senator Zeldin and members of his panel, recently traveled to Albany to provide testimony to the Senate Mental Health committee on their findings. Following this hearing, Senator Zeldin worked with Senator Roy McDonald (R, C, I- Wilton), chairman of the Mental Health Committee, to include funding for the PFC Joseph Dwyer Program in the 2012-2013 General State Budget.
How a Bill Becomes LawLearn More
Senator has new policy idea
Idea is drafted into a Bill
Bill undergoes committee process
Senate and Assembly pass bill
Bill is signed by Governor


text:
Rebecca Minkoff Women's Roxanne Ankle-High Suede Pump
- Fashion Trend: Ankle-High Suede Pump
Special Price $136.95 was $228.00
In stock
These Rebecca Minkoff Roxanne's are guaranteed to add a flare of style to any wardrobe. The 4.25" inch heel and 2.5" inch shaft will have you looking like you are ready to walk the runway where ever you may be. This stiletto also features a open toe for supreme comfort while looking your best.
Write Your Own Review


text:
California High School Physics Standards
Descriptions of basic demonstrations in Word format that align to each of the CA high school physics standards. There are pictures of the demonstrations along with a short explaination. Most are a page or two in length featuring a couple of demonstrations that can be done with easily found materials.
Material Type: Collection
Technical Format: HTML/Text
Date Added to MERLOT: June 20, 2009
Date Modified in MERLOT: June 20, 2009
Submitter: Kathryn Beck
Reviewed by members of Editorial board for inclusion in MERLOT.Editor Review (not reviewed)
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- Bookmark Collections (1)
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Primary Audience: High School
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text:
It has been quite a while since I did the first installment of this series, but here are a few more items to add to the "favorites" pile!
1. Nars Blush in Gaiety: This is a cool-toned, pink blush that completely brightens up a complexion! It's somewhat frightening in the pan, but looks incredibly natural on the skin. After making my blush rounds through the cosmetic counters (so to speak), I've decided that Nars has incomparable quality. It lasts long, applies beautifully, and has pretty packaging! (Always a plus!) I purchased it about a month ago and have been using it almost every day, along with my next favorite...
2. Nars Blush in Orgasm (I hate that they chose this name!): I've purchased this before, but was less than thrilled. However, I've recently come back to it and can't understand how I ever passed it up. This is the prettiest coral shade, with more peachy/golden tones than pinks. It gives you a subtle sunkissed glow & should absolutely be a staple in every girl's makeup bag!
3. Benefit Hoola: I've tried a TON of bronzers. Estee Lauder, Nars, Bobbi Brown, MAC, Clinique, Lancome, Chanel, Smashbox, Too Faced ...I can go on and on. This is by far my favorite. It's the perfect shade for my skin in which to add contour. I place it under my cheekbones, along my jawline, on my temples ...then kind of, well, blend it a bit all over where the sun naturally hits my face. I hate the bulkier packaging, but the truth is that I feel like a puffy fish-face without it. Plus, it's a better value than other brands at $28 for .40oz.


text:
Border Scramble
|Roll sausage
|1 Pound (Original Recipe)
|Chopped cooked potatoes
|1 1⁄2 Cup (24 tbs)
|Chopped onions
|1 1⁄2 Cup (24 tbs)
|Chopped tomatoes
|1 1⁄2 Cup (24 tbs)
|Chopped green bell pepper
|3⁄4 Cup (12 tbs)
|Picante sauce
|1⁄2 Cup (8 tbs)
|Hot pepper sauce
|1 Tablespoon
|Garlic powder
|1⁄2 Teaspoon
|1⁄2 Teaspoon
|9 inch flour tortillas
|Prepared meatless chili
|2 Cup (32 tbs)
|Shredded cheddar cheese
|2 Ounce (1/2 Cup)
Crumble sausage into large skillet.
Cook over medium heat until browned, stirring occasionally.
Drain off any drippings.
Add all remaining ingredients except tortillas, chili and cheese; simmer 20 minutes or until vegetables are crisp-tender.
To warm tortillas, microwave 1 minute at HIGH between paper towels.
Place 1 cup sausage mixture in center of each tortilla; fold tortilla over filling to close.
Heat chili in small saucepan until hot, stirring occasionally.
Top each folded tortilla with 1/2 cup chili and 2 tablespoons cheese.
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 2881 Calories from Fat 1490
% Daily Value*
Total Fat 166 g255.3%
Saturated Fat 57.9 g289.7%
Trans Fat 0 g
Cholesterol 340.8 mg113.6%
Sodium 9112 mg379.7%
Total Carbohydrates 250 g83.5%
Dietary Fiber 44 g175.9%
Sugars 44.8 g
Protein 133 g266.7%
Vitamin A 158.3% Vitamin C 258.1%
Calcium 78.5% Iron 154.6%
*Based on a 2000 Calorie diet


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Lyrics.com »
Search results for 'last time by oar'
Yee yee! We've found 10,893 lyrics, 100 artists, and 100 albums matching last time by oar.
Part Time Party Time Band · Time After Time · The Last Poets · Last Nickel · The Last Resort · Last Gentlemen · Last Tribe · James Last · James Last & His Orchestra · Last Real Texas Blues Band Featuring Doug Sahm – and 90 other artists »
More Oar: A Tribute to Alexander "Skip" Spence (Hutchinson, Hutch) · The First Time... For the Last Time (Shania Twain) · Time Time Time: The Complete UK Singles (And More) 1965-1967 (The Knack) · Every Last Time (Gameface) · Last Time (Moderat) · The Last Time [Bonus DVD] (John Farnham) · Last Time (Moderat) · Foiled for the Last Time (Blue October) · One Last Time (Gavin Mikhail) · This Is the Last Time [UK CD] (Keane) – and 90 other albums »
some more By the way, is there any you can sell us? Time out, save the play Trying to make the lie last Any old way we can Signed out all
Watching as it flies by (Spy the unknown) Focus on the quiet oars Tell me how far you'd go (Go where you like) Knowing your air won't last (Last one to know)
All in the golden afternoon full leisurely we glide For both our oars, with little skill, by little arms are plied While little hands make vain
at the oars G#7 C#m Or staring from the rail E -A B7 And the sea bids farewell G#m She waves in swells
at the oars G#7 C#m Or staring from the rail E -A B7 And the sea bids farewell G#m She waves in swells
bay And my crew are the bravest ever handled an oar And not by the tempest, they laugh at its roar No ballroom can tempt me nor raise my despair For
to a place A land that's not so far away The boat above our heads for the walk to the shore As feelings of dread soak the length of the oar Connecticut
Once I tried to row 'Cause you know I hate to fly so I set out from the coast But got battered by the tide As I glided back to shore I raised
Movin' in next door, searchin' from floor to floor You know what they're lookin' for Someone's always keepin' score every time you dip the oar Love
Pulling the weight up against the wind Is the plight of the galley slave Chained to this cold bench, six to the oar Sentenced to an early grave
Slow your lovin' up Slow your lovin' down Love gaining not losin' ground No worry No hurry Take your sweet time Baby. Dabble dally Shilly
The sound of the earthquake, The rhythms that you make, Are makin' my head shake in two. Magnetized by you Your love will pull me through
at them with force All men to the oars Row for all your worth Most likely this will be your last day On this wretched earth The weak they try
sold It's value in the past ten years Has increased twenty fold I hope the man Won't change too much Oh mister owner please I'd love to drive by one more
And all that time, i felt just fine. I held do many people in my suitcase heart that i had to let the whole thing go. It was taken by the wind and snow,
One by one Every courtesy is gone And the show begins Bring on the clowns Pain is quiet I've laughed at silence For the last time Let the show
of the fifth day Before the sun arose They hear bells chime and see pyres Torched at a nearby coast "LOWER THE SAIL, GRAB YOUR OARS, NOW MEN IT'S TIME
Nothing lasts forever, you should know that by now Good times, heartache You'll get through this trouble though you may not know how Your heart won't
all of their problems on your plate? All your good friends Who can see the real you? All your old friends Who sticks their oar in everything you
the last booth left That the rest of us avoided cause the bathroom stench On the bench there's some papers with the local fare There's a chicken that I
Last time Last time I saw Jeannine She looked just like a royal queen And she cruised by With some wealthy guy In a Cadillac limousine Last
weak, my love is blind When your game is up, I'll still be by your side If you love me at all, let this be the last time That you break my heart That you
"And now for something completely different" Hey!, Fuck you we crush crews the oar way I'm, Sicker then using period blood for war paint Fans
Time Time Time Forever time It's time to walk by me One last time, it's time At first Relax Get set Get your message from time Time, time, time, time
wealth and fame," Promised great bounty by the blessings of Njord. "Vast wealth awaits you in the land across my seas." Wind fills the sail, oars pound


text:
Windows Phone Calculator
- Status Closed
- Budget $50 - $250 USD
- Total Bids 19
Project Description
Hello developers,
I need someone to build me an scientific calculator app for Windows phone. Program language must be C# (Silverlight). The app should support WP7 and WP8 and should have all features and functions like classic sci-calculators. The design layout for this app is in the attachment. We'll consider only bids (PM's) which contain a short description where you explain which components will you use. (This way we'll filter real bidders from fake ones). Thanks!Get free quotes for a project like this
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text:
I love summer produce! The fruits are sweet and the veggies... yum! After picking up 25 pounds of peaches at our local food co-op, I was on the hunt for innovative ways to eat them (because as much as I would have liked, peach cobbler was not an acceptable choice for each of my 3 daily meals). I came across a recipe for peach and watermelon salsa served over tomatoes, and I was intrigued. So I got to work, tweaked it a little, and came up with a quite yummy recipe: Spicy Peach and Watermelon Salad over Fresh Tomatoes. I'm drooling just thinking about it.
Here's how it goes (if you want to skip to the recipe, just scroll down!):
First, in a medium to large bowl, mix together 1 tablespoon lime zest, 1/4 cup lime juice, and about 1/2 cup of your favorite fruity jalepeno jelly. I used Raspberry Jalapeno jelly, and it turned out delicious.
Prepare all of your fruit...
Cube 1/2 small watermelon. TIP: Cut your watermelon half into two slices. With rind still on, cut slices through the watermelon both parallel and perpendicular to your body, making about 1" x 1" squares. Then, run your knife between the rind and the flesh to cut off the rind, leaving cubed pieces! Perfect!
Peel, pit, and cube 3 peaches. If you read my recent recipe post on the Mini Pavlova with Peaches and Blueberry Sauce, I shared this little tid bit on peeling peaches:
Next, add your watermelon, peaches, and just a smidge (less than 1/4 cup) shredded basil to your pepper jelly mix. Toss to coat well.
Slice a ripe organic vine-ripened tomato and put slices in a small bowl or cup. Add your spicy peach and watermelon salad over your tomatoes. Garnish with a sprig of basil for presentation.
She's a beauty, ain't she?
Now, to summarize, here's the full recipe:
- 1/2 cup spicy fruit and pepper jelly (ie Raspberry Jalapeno Jelly)
- 1 tablespoon lime zest
- 1/4 cup lime juice
- 2 cups cubed seedless watermelon
- 1 cup peeled, pitted, and cubed peaches
- <1/4 cup shredded basil, fresh
- 3-5 vine-ripened tomatoes, sliced into bite size pieces
- Mix together hot fruit and pepper jelly, lime zest, and lime juice.
- Add in watermelon, peaches, and basil. Toss well to coat.
- Put tomatoes in bottom of small bowl or cup. Top with Spicy Peach and Watermelon salad mix.
- Enjoy!
Looking for more peach recipes?


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A significant role for increased platelet reactivity in the pathophysiology of occlusive arterial thrombosis is widely recognized. However, the mechanisms responsible for enhancing platelet reactivity in vivo during hyperlipidemia are poorly understood. We have recently isolated and structurally defined a novel family of oxidized choline glycerophospholipids (oxPCCD36) that are present in vivo at sites of enhanced oxidative stress. oxPCCD36 serve as high affinity ligands for scavenger receptors class B and modulate platelet reactivity and thrombosis in oxidative stress. Our preliminary studies demonstrated that a class of products formed as a result of degradation of oxPCCD36 by PLA2 (carboxyalkylpyrolle protein adducts) is a new and powerful platelet agonist, however, the receptors mediating its effect are not known. Platelets express a number of receptors with pattern recognition properties, including several toll like receptors (TLRs). A recent study suggested that platelet TLRs may modulate thrombosis when their ligands are present in circulation. This proposal will address the hypothesis that platelet TLRs alone, or in cooperation with scavenger receptors modulate platelet reactivity and prothrombotic state induced by endogenous ligands generated in oxidative stress. This proposal will also pursue the hypothesis that carboxyalkylpyrolle protein adducts serve as novel ligands for platelet scavenger /toll like receptors and identify the mechanisms of prothrombotic activity. Finally, we will continue the investigation of the molecular mechanism of scavenger receptor BI regulation of platelet function and thrombosis in dyslipidemia. Our preliminary data strongly support our hypothesis.
The Specific Aims are:
Aim1 : To assess whether the platelet activating and prothrombotic activities of oxPCCD36 are mediated by platelet TLRs alone, or in cooperation with scavenger receptors class B.
Aim2 : To investigate the role of scavenger receptor-BI in platelet function in dyslipidemia and oxidative stress.
Aim3 : To elucidate the mechanism and assess the physiological and pathophysiological consequences of the interaction between carboxyalkylpyrolle protein adducts (CAPs) and platelets.
Risk for platelet-mediated blood clotting is increased in atherosclerotic disease; however, the mechanisms are poorly understood. We have recently demonstrated that specific oxidized phospholipids can control platelet function via platelet receptors. In this proposal we will study how platelet toll like receptors regulate platelet function and thrombosis in atherosclerosis.
|Zimman, Alejandro; Titz, Bjoern; Komisopoulou, Evangelia et al. (2014) Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin. PLoS One 9:e84488
|Gao, Detao; Willard, Belinda; Podrez, Eugene A (2014) Analysis of covalent modifications of proteins by oxidized phospholipids using a novel method of peptide enrichment. Anal Chem 86:1254-62
|Panigrahi, Soumya; Ma, Yi; Hong, Li et al. (2013) Engagement of platelet toll-like receptor 9 by novel endogenous ligands promotes platelet hyperreactivity and thrombosis. Circ Res 112:103-12
|Somanath, Payaningal R; Podrez, Eugene A; Chen, Juhua et al. (2011) Deficiency in core circadian protein Bmal1 is associated with a prothrombotic and vascular phenotype. J Cell Physiol 226:132-40
|West, Xiaoxia Z; Malinin, Nikolay L; Merkulova, Alona A et al. (2010) Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands. Nature 467:972-6
|Ma, Yi; Ashraf, Mohammad Z; Podrez, Eugene A (2010) Scavenger receptor BI modulates platelet reactivity and thrombosis in dyslipidemia. Blood 116:1932-41
|Zimman, Alejandro; Podrez, Eugene A (2010) Regulation of platelet function by class B scavenger receptors in hyperlipidemia. Arterioscler Thromb Vasc Biol 30:2350-6
|Podrez, Eugene A (2010) Anti-oxidant properties of high-density lipoprotein and atherosclerosis. Clin Exp Pharmacol Physiol 37:719-25
|Gao, Detao; Ashraf, Mohammad Z; Kar, Niladri S et al. (2010) Structural basis for the recognition of oxidized phospholipids in oxidized low density lipoproteins by class B scavenger receptors CD36 and SR-BI. J Biol Chem 285:4447-54
|Malinin, Nikolay L; Zhang, Li; Choi, Jeongsuk et al. (2009) A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans. Nat Med 15:313-8
Showing the most recent 10 out of 17 publications


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In Racial Ethnic & Women's Ministries, we engage the Church in its mission to become more diverse and inclusive of racial, ethnic, cultural and language groups, and we equip women for leadership in all ministries of the Church. We are blessed to see a vision of Pentecost every day. There are Presbyterian Mission Agency offices supporting African, African American, Asian, Hispanic/Latino-a, Korean, Middle Eastern, Native American and Portuguese-language emerging ministries. The racial ethnic and new immigrant worshiping communities that we support regularly gather together, constructing a racial ethnic identity that stems from a common history, heritage and language. We work closely with racial ethnic and new immigrant worshiping communities through mid councils, caucus groups, and networks, engaging in the mission to grow, transform, empower, lead and develop communities. We create an awareness of institutional racism in the church and an environment where racial ethnic persons, new immigrants, and women can serve fully in ministry and leadership.
In our ministries we work to achieve gender and racial justice and equip racial ethnic women, men and young adults for leadership. We assist college-age and racial ethnic young women in their faith journeys and foster networks of support for them.
As we discover how to share the gospel in an increasingly diverse culture, we have a greater chance of living out the Biblical vision of a world where the humanity of everyone is valued and where God's love is spread to every race, class, culture and people.
We Serve a Messiah Whom We Celebrate, Treasure and Adore
"So [the shepherds] went with haste and found Mary and Joseph, and the child lying in the manger. Whey they saw this, they made known what had been told them about this child; and all who heard it were amazed at what the shepherds told them. But Mary treasured all these words and pondered them in her heart." —Luke 2:16–19 (NRSV)
When Mary and Joseph had their child in a stable with animals all around, and the only crib that this poor, migrant couple found available was a trough where the animals fed, then you'd expect that they would be filled with despair. But instead, I imagine that this was one of the happiest moments of their lives. The word spread that this special baby was born. Rich and poor came to see him. Young and old peered into the manger. Race, gender and class distinctions went away when the diverse people of God came to pay their respects to the child, who would become the messiah of the world!
Our savior Jesus Christ was born in a time of anxiety and uncertainty. Today's climate seems similar. The newest confession in the Presbyterian Church (U.S.A.)—The Confession of Belhar—is encouraging during this time in our history. It calls the church to "witness against and strive against any form of injustice. . ." Belhar also calls us to:
Reconciliation and Building Up God's people
"We believe that Christ's work of reconciliation is made manifest in the church as the community of believers who have been reconciled with God and with one another; that unity is. . . one which the people of God must continually be built up to attain."
A New Humanity and Suffering with One Another
"We believe. . . that we need one another and upbuild one another, admonishing and comforting one another; that we suffer with one another for the sake of righteousness."
Celebrate Various Languages and Cultures
"We believe. . . that the variety of spiritual gifts, opportunities, backgrounds, convictions, as well as the various languages and cultures, are by virtue of the reconciliation in Christ, opportunities for mutual service and enrichment within the one visible people of God."
Reject the Separation of People on A Racial Basis
"We believe that the credibility of this message is seriously affected and its beneficial work obstructed when it is proclaimed in a land which professes to be Christian, but in which the enforced separation of people on a racial basis promotes and perpetuates alienation, hatred and enmity; that any teaching which attempts to legitimate such forced separation by appeal to the gospel, and is not prepared to venture on the road of obedience and reconciliation, but rather, out of prejudice, fear, selfishness and unbelief, denies in advance the reconciling power of the gospel, must be considered ideology and false doctrine.
"In times of anxiety, we may be tempted to despair, but our faith encourages us to be hopeful, for we serve a messiah whom we celebrate, treasure and adore.
"We believe that, in obedience to Jesus Christ, its only head, the church is called to confess and to do all these things, even though the authorities and human laws might forbid them and punishment and suffering be the consequence. . . To the one and only God, Father, Son and Holy Spirit, be the honor and the glory for ever and ever."—Excerpts are from The Confession of Belhar
Christmas Blessings,
Rhashell D. Hunter
Read the Racial Ethnic Torch
In the current issue of the Racial Ethnic Torch, we lift up the work that Racial Ethnic & Women's Ministries, and the Presbyterian Church (U.S.A.) as a whole, is doing to overcome racism. Some of the articles featured in this issue include:
- "Facing Racism: A Vision of the Intercultural Community"
- "Churchwide Conversation on Race, Ethnicity, Racism, and Ethnocentricity"
- "Women of Faith": 2016 Award Recipients


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As per Thomson Reuters, Advance Auto Parts Inc (NYSE:AAP) stock can hit $161.94 levels in upcoming months. It is the price acknowledged after weighing renowned analysts' calls.
The unit follows the idea of assessing reports to ascertain price target. Dependent on poll, the firm probable EPS for quarter is $1.15 while it is $7.43 for current fiscal.
Valuation Level
Market analyst, immaterial of the brokerage houses, repeatedly use price-to-earnings ratio for purpose of Advance Auto Parts Inc Advance and other firm's valuation. Without even knowing what is the financial worth of a company or how it fundamentals are, traders are inclined to forecast the right time to enter any trade whether it is a buy or short-sell. Market participants can lose out capitalizing opportunities if the investment choices are taken entirely on equity's existing market value. It should be recognized that many means are there to gauge the firm performance. As a result, investors allay their odds of liquidating stocks too soon and losing future profits or spending in an equity appraised too high set against its actual worth. Advance Auto Parts Inc (NYSE:AAP) P/E ratio is 23.92 while Price-to-Earnings-Growth ratio is 1.89.
Technical Forecast
While reviewing Advance Auto Parts Inc Advance on the technical element, the conclusion was share is trading $-4.76 points away or -3.12% from 50-day MA of $152.89. It is $-8.84 or -5.63% away 200-day moving average of $156.97. MA designates for moving average.
Last year Advance Auto Parts Inc (NYSE:AAP) stock made a high of $201.24. The lowest level was $131.59. If stock jumps over $-53.11, there will be a 52-week high. However, when stock drops $+12.57% points, investors should embrace 52-week low.
An equity's price fluctuations throughout a session changes as per the demand and supply. The market price assists buyers find whether the capitalizing opportunity in shares. The price of equity turns out to be dynamic when implementing trading plans. For illustration, buyers have a substitute option to place orders. This can lessen financial losses or assist investors to pull market gains.
1 Chart Pattern Every Investor Should Know
This little-known pattern preceded moves of 578% in ARWR, 562% in LCI, 513% in ICPT, 439% in EGRX, 408% in ADDUS and more...


text: Go ahead and share the bed with your dogwe've got it covered. Our lightest dog-proof bedding ever, designed to stay in place to protect your bedding from dirt, pet hair, and moisture, but not weigh you down when it comes to washing. This reversible coverlet has a sturdy polyester corduroy weave on one side and a water-resistant coating in camel on the other side. Matching shams are all corduroy with envelope-style opening on back. Shams sold separately.


text:
Call (888) 651-9785
January 2017
Shame there was no wind but the crew more than made up for that. Beautiful boat and location. Skipper let me steer some way into harbour which was a hoot!
November 2016
This was the perfect way to see a Barcelona sunset. Our captain was entertaining, knowledgable and we all shared a few stories and laughs along the way. It really did feel like we were on our own yacht for the evening. Highly recommended.
October 2016
If we could give more than 5 stars we would! This was a definite highlight of our trip to Barcelona! It was supposed to be a small group, but it ended up being just myself, my brother, and my mother. Our guide was so friendly and accommodating - it could not have been more perfect!
October 2016
Frank was wonderful and accommodating during our Mediterranean tour. He was very personable and taught us a lot about the history of Barcelona. It was very beautiful and I STRONGLY recommend this tour!
September 2016
This was an awesome tour with a great tour guide. Cruising the gorgeous sea was breathtaking and getting a chance to dip into the water was fun. I highly recommend!
September 2016
The tour was amazing, we had a chance to see stunning views of Barcelona from the sea. The skipper was extremely friendly, and provided us with knowledge about Barcelona. I would highly recommend this trip.
twills Australia
September 2016
Wow. Everything went perfectly to plan. Our captain met us exactly on time and we set sail into the Med and travelled along the Barca coast for a good two hours. Gorgeous waters turning from Emerald green to a perfect blue once out of the docks area. The captain knew his stuff and was a pleasure to talk to. He genuinely enjoyed taking us out. It was more than a job to him. We were fortunate in it was just the two of us on the boat but it's a maximum of 6 anyway so it's still personal.
August 2016
Great afternoon on Med! Nice boat! Nice crew! Great weather!
Darybel O United States of ...
August 2016
Great time during this hour and a half sailing trip from Barcelona, the sail boat captain was very friendly and made sure I was comfortable all the time. He is very knowledgeable and extremely friendly! I highly encourage anyone going to Barcelona to take this trip, the views of Barcelona coast line are amazing!
June 2016
Everything it was promised to be!!
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Short sale. 4 bedroom / 2.5 bathroom Colonial home w/ 1-car garage plus driveway parking on a 35' x 100' lot. Directly across from Elmwood Park. Cash or 203K (renovation loan) buyers only. All offers are subject to lien-holder / 3rd party approval. Buyer is responsible for all municipal permits and certificate required to close. This is an "as-is" sale.
Listing Courtesy of KELLER WILLIAMS - NJ METRO GROUP


text:
NORMAN, Okla. -- Romero Osby is hoping for a repeat performance.
Oklahoma's junior forward knows what it is like to be on a team that gets on a tear to end the season and earns an NCAA Tournament berth by winning its conference tournament.
Osby was a freshman when Mississippi State won the 2009 SEC Tournament, winning six straight games -- including four straight SEC Tournament contests -- after going 1-5 in its six previous games. Osby played 12.6 minutes per game, averaging 4.1 points and 2.6 rebounds for a Bulldog squad that finished 23-13 overall that season. He transferred to OU after his sophomore season.
Now, as one of the leaders of the Sooners, Osby is hoping to use that experience to help his teammates understand their postseason destiny remains under their control despite a lackluster record (13-13, 3-11 in Big 12).
"We were just like this team," Osby said of the 2008-09 Bulldogs. "Up and down, couldn't really get a road win when we needed it, couldn't get a home win when we needed it.
"This team has a lot of similarities."
With the Sooners in the midst of a six-game losing streak and needing to go on a winning run for any hopes of postseason play, Osby reflected on how the Bulldogs did it.
"Everyone's mindset was, 'We're not going to be selfish, we are going to be together and play defense,' " he said. "And we came together and won that tournament."
Struggles with trying to do too much as individuals has been part of OU's problem this season. When opponents go on scoring runs, the Sooners allow the runs to take them out of their rhythm and begin trying to do things individually -- either on the offensive or defensive end -- leading to turnovers and open shots for the opposition.
That's one of the main differences between the two teams in Osby's eyes.
"Not saying we are being selfish, but everybody forgot about themselves [at MSU]," Osby said. "I feel like we can do the same thing, if we do that ... just be as unselfish as possible on both ends."
They'll also have to start learning from their losses.
OU repeatedly has seen teams pull away from it in the second half, and the Sooners still haven't learned from it with the end of the regular season quickly approaching. Iowa State became yet another team to pull away in the second half and defeat the Sooners on Saturday. The Cyclones outscored OU 33-12 during a 10-minute stretch in the middle of the second half.
"Sometimes you have to remember what happened last time," Osby said. "You have to think about it when you are on the floor, because it has happened to us in pretty much every game we've lost."
Osby thinks his team has learned from its 11 conference losses, but they aren't translating what they have learned to the next game.
"It has to be everybody; it can't be one or two people thinking about that."
Nonetheless, the Sooners forward continues to have postseason dreams despite a disappointing season, because he's been on a team that has done it before, and he is confident his team will learn, and get better, from all of the losses it has endured since January.
"It's the perfect time to make a run and earn our way into the NCAA Tournament or earn our way into the NIT Tournament," Osby said.
Brandon Chatmon covers University of Oklahoma sports and recruiting for SoonerNation. He can be reached at firstname.lastname@example.com.


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UEIUA CMP - 2420 PWM 20A 12V / 24V Solar Charge Controller
Main Features:
● Uses PWM charge mode, which can improve system efficiency and prolong the lifespan of the battery
● Uses single chip microcomputer control
● Clear charge state, discharge state, and battery power display
● Adopts the modified shell which accords with air flow, has better cooling effect ( compare with the old version )
● With overcharging protection, lightning protection, under voltage protection, overload protection, short circuit protection and reverse polarity protection
|Specification parameter
|Weight and Size
|Package Contents
We do not accept any responsibility or liability for misuse of this or any other product. All our products are extensively tested to comply with rigorous and strict QC standards. For certain products (e.g. toys, knives, etc.), we recommend proper supervision as we cannot be held liable for misuse or accidents.
Inquiry Question


text:
ACHICA > Collarways > Flexi Funtime Mini Tape Dog Lead, Pink
Click image to zoom -- or click here to enlarge
Flexi Funtime Mini Tape Dog Lead, Pink
This product is not available
|Sale closed
UK Delivery: Estimated 27th Oct - 1st Nov - we'll email you once your items have been dispatched.
Returns: Click Delivery & Returns or check your delivery confirmation.
If you are ordering multiple items, ACHICA aim to send as few deliveries as possible for your convenience – you will only pay one delivery charge.
|Product Information
Colour: Pink
Product Dimensions: Mini 3m cord length.
For dogs up to 12kgs
Product Code FLFTXSP
|Delivery & Returns
We hope you will be thrilled with your purchases but in the unlikely event that you want to return something, you can find full return instructions on the back of your delivery note or click here


text:
Top Definition
Kids who dont belong on the face of the earth and appear on birth control commercials
Eric Fishel should be a Birth Control Poster Boy because hes so fuckin ugly and half-retarded looking
by True to my Roots July 07, 2003
The Urban Dictionary Mug
One side has the word, one side has the definition. Microwave and dishwasher safe. Lotsa space for your liquids.Buy the mug
The Urban Dictionary T-Shirt
Smooth, soft, slim fit American Apparel shirt. Custom printed. 100% fine jersey cotton, except for heather grey (90% cotton).Buy the t-shirt


text:
Be sure to check out my other site Gym Free Fitness if you're looking for information on how to eat healthy and stay in shape at home. The site focuses on cutting through all the BS and bringing you only helpful information. You won't find any quick fixes, detox pills, or juice cleanses over there... though I do love a delicious … [Read More...]
The One Blog Customization You Should Do Today
A few months ago I was reading an article on someone's blog and decided to leave a comment. Doesn't sound too out of the ordinary right? But what happened next was … [Read More...]
Snag My Behind the Scenes Blog Checklist
Let's face it, there is a lot more involved in having a blog than just writing posts and sharing them on social media. If you've been blogging for any length of time … [Read More...]
How to Find Free Pictures to Use on Your Blog
It's a problem that a lot of bloggers have... finding images to use on your blog that won't break the bank. If you've been searching for the perfect resource for … [Read More...]
What are the Best Times to Post to Facebook?
If you're a blogger, you have undoubtedly seen the most recent video that Holly Homer did with Crystal and Company regarding growing your Facebook page likes. In it, … [Read More...]
How to Make Eggnog in Just 20 Minutes
Like many people, one of my favorite things about the holiday season is eggnog. But what I don't like is store bought eggnog and all the extra ingredients and … [Read More...]
No Fuss 1 Hour Recipe for Thanksgiving Turkey
I think most of us remember our mom getting up early on Thanksgiving morning and getting the turkey into the oven, then spending all day checking and basting the … [Read More...]
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Tri County Preschool
Tri County Area Schools is pleased to be able to offer several preschool options to parents.
Great Start Readiness Program
The Great Start Readiness Program (GSRP) is a free preschool program funded through the State of Michigan for students who meet eligibilty requirements.
Tri County Preschool
Tri County also offers a tuition based preschool for families who do not qualify for GSRP, as well as a 3 year old program, both free and tuition based.
Head Start Program
Edgerton also houses the Head Start program provided through EightCAP, which is a federally funded preschool for 3 and 4 year olds, based on income.
Only one application is necessary to determine which program your child may qualify for.
GSRP & 4 Yr Old Preschool Info
3 Yr Old Preschool Info
Thurs. Sept 1 - 4yr Preschool Open House 5:30 - 6:30 pm
Tues. Sept 6 - 4 yr Preschool First Day of School
Thurs. Sept 8 - 3yr T/Th Preschool Open House 9:30 - 10:30 am
Fri. Sept 9 -3yr W/F Preschool Open House 9:30 - 10:30 am
Tues. Sept 13 - 3yr T/Th Preschool First Day of School
Wed. Sept 14 - 3 yr W/F Preschool First Day of School