Design Of Sarda Type Fall Pdf Download REPACK
We first extracted bibliographic information from the articles, this included the date of publication, the country where the research was conducted, the sample size and demographics of the sample (i.e., mean age, gender, and cultural or ethnic background), the domain of application, the design of the study, and the name, design, and capabilities of the social robot. The type of outcome (categorised as general attitudes, affective attitudes, cognitive attitudes, behavioural attitudes, trust, anxiety, or acceptance) and details of the measures used to assess each outcome (e.g., the NARS) were identified and extracted next.Footnote 2
Design Of Sarda Type Fall Pdf Download
An adaptive DNA sequencing strategy was developed to determine the 771 bp PRNP coding sequence for any sheep and thereby produce a consensus sequence for targeted flocks. The strategy initially accounted for 43 known polymorphisms and facilitates the detection of unknown polymorphisms through an overlapping amplicon design. The strategy was applied to 953 sheep DNAs from multiple breeds in U.S. populations. The samples included two sets of reference sheep: one set for standardizing PRNP genetic testing and another set for discovering polymorphisms, estimating allele frequencies, and determining haplotype phase. DNA sequencing revealed 16 previously unreported polymorphisms, including a L237P variant on the F141 haplotype. Two mass spectrometry multiplex assays were developed to score five codons of interest in U.S. sheep: 112, 136, 141, 154, and 171. Reference tissues, DNA, trace files, and genotypes from this project are publicly available for use without restriction.
First, a set of tissues was assembled from 21 healthy sheep representing all diplotype combinations of the six most common haplotype alleles at positions 136, 154, and 171 (i.e., ARQ, ARR, AHQ, ARH, VRQ, and ARK; Table 2). Second, tissues of three additional sheep were included to represent the diplotype combinations of codon 112 (i.e., MM, MT, and TT) that occur on the ARQ haplotype. Third, tissues from sheep with four of six possible diplotype combinations of the three haplotype alleles known for codons 141 and 237 (i.e., haplotype alleles LL, FL, and FP) that occur on the ARQ haplotype. The variants of the ARQ haplotype were included because alleles at positions 112 and 141 have been implicated in scrapie resistance and are available in our populations. The two remaining tissue sets needed to complete this collection are expected to be produced in the spring of 2010 and available in the fall (i.e., MALRQL, MAFRQP and MAFRQL, MAFRQL; Table 2). Approximately 2 to 3 kg of DNA-rich tissues were collected from each animal sampled, thus providing a significant supply for wide-spread use. The complete PRNP coding sequence has been determined for each of the 28 animals and deposited in GenBank (Table 2). In addition, a set of 20 highly informative autosomal ovine SNPs were scored to provide a genetic "bar code" for tracking these samples within and between laboratories and resolve sample mix-up issues where they occur (Table 2).
A 314 bp PRNP fragment was amplified from genomic DNA for scrapie susceptibility testing. In addition to scoring the widely implicated codons 136, 154, and 171, our assay was designed to score codons 112 and 141 to facilitate investigation of these alleles in scrapie infected flocks. The 314 bp fragment had no known polymorphisms in its amplification primer binding sites (Figure 1C). Accurate codon diplotype scoring for multiple adjacent SNPs was achieved in two reactions where both the sense and antisense DNA strands were simultaneously scored in the same reaction. Codons 112, 136, and 154 were scored in one multiplex reaction, whereas codons 141 and 171 were scored in another (Figure 4). Although T136 was not present in the sheep tested, a synthetic DNA control for T136 produced good results when added to DNA amplified from homozygous A136 sheep. For codons 112, 136, 141, and 154, scoring from either DNA strand produced a complete diplotype. Thus, when both DNA strands were scored in the same reaction, concordance provided an internal diplotyping control. This was important because 15 other nearby SNPs were known to be present in eight of ten extension primer binding sites and may cause allele dropout in certain animals (Figure 1C). Scoring codon 171 required analysis of both sense and antisense strands to unambiguously infer the diplotype (Figure 4B, D, and 4J). In blind comparisons between diplotypes derived from Sanger sequence versus those from hME MALDI-TOF MS, 100% concordance was observed for the 28 sheep from the Scrapie Control Panel and the 192 parents from the Diversity Family Panel (data not shown). Together, these hME assays provide one example of well-characterized high-throughput MALDI-TOF MS assays for scoring PRNP codons 112, 136, 141, 154, and 171.
Mass spectrograms of ovine PRNP codons at positions 112, 136, 141, 154, and 171. A single PCR reaction was used to amplify a 336 bp genomic DNA region and the product split for use in two subsequent multiplex hME reactions. Spectral peaks represent singly-charged ions whose mass-to-charge ratio (m/z) was compared with calibrants for mass determination. Spectra feature labels: s and a, sense and antisense analytes produced from respective hME extension primers; p, unincorporated extension primer; , peak height clipped to conserve space. Two artifact peaks are produced as a consequence of multiplex design considerations. The first is a g nucleotide "pausing peak" in the codon 141 antisense assay (5530 Da, feature label "1"). The second artifact peak (feature label "2") is a g nucleotide misincorporation/insertion followed by a ddT termination in the codon 141 sense assay, i.e. 5'-[primer]-CGddT-3' (4866 Da). The correct termination product is 5'-[primer]-CddT-3' (4537 Da). This artifact peak at 4866 Da appears sporadically and independent of sample type or quality. Panels A and B: mass spectrograms illustrating the A136V and Q171R heterozygote. Panels C and D: mass spectrograms illustrating the R154H and Q171H heterozygote. Panels E and F: mass spectrograms illustrating the L141F heterozygote. Panels G and H: mass spectrograms illustrating the M112T heterozygote. Panels I and J: mass spectrograms illustrating the A136T and H171K heterozygote. The T136 was a synthetic allele that was added to the primer extension reaction cocktail to reference animal 200665213 (homozygous for A136).
A group of diverse rams were mated with ewes to produce families with twin lambs (i.e., tetrad families, Figure 2). Autosomal SNP diplotypes at 60 SNP loci were used to confirm relationships among sheep from 96 candidate tetrad families. These SNP loci included five from PRNP and 55 at other sites distributed across the genome (Additional File 1). Analysis of the 60 MALDI-TOF MS diplotypes for all 96 candidate families (i.e., 23,040 diplotypes) showed that Mendelian inheritance patterns were present in 94 of 96 families. Two families each had a single non-Mendelian inheritance pattern attributed to a distinct SNP. However, subsequent diplotypes scored from redundant Sanger sequencing revealed that the two MALDI-TOF MS diplotypes were incorrect. This error rate (two detected errors per 23,040 scored diplotypes) is well within the 99% accuracy expected for multi-plexed MALDI-TOF MS diplotype scoring and thus, the proposed family relationships in all 96 tetrad families appeared to be correct. The diverse group of sires for these families represents a minimal set of sheep and breeds for SNP discovery and allele frequency estimation. Their dams and offspring allowed haplotype phasing and verification of rare SNPs by allele segregation, features important for designing efficient and accurate DNA tests.
Three sequential versions of USMARC Sheep Diversity Panels were used. The purpose of these panels was SNP discovery and allele frequency estimation. The first panel version (1.1, ) consisted of 90 rams from nine breeds (Dorper, White Dorper, Dorset, Finnsheep, Katahdin, Rambouillet, Romanov, Suffolk, and Texel) and a composite population (USMARCIII: 1/2 Columbia, 1/4 Hampshire, and 1/4 Suffolk ). These breeds were selected to represent genetic diversity for traits such as fertility, prolificacy, maternal ability, growth rate, carcass leanness, wool quality, mature weight, and longevity. The ten rams sampled from each breed were chosen to minimize genetic relationships among rams within breed. The second version (2.0) consisted of 96 rams from nine breeds and the composite population and was based on the same design as version 1.1. However, version 2.0 contained 78 rams not present on version 1.1. The third version (2.4) consisted of 95 rams from nine breeds and the composite population, plus one Navajo-Churro ram with a rare prion haplotype allele (ARK). The version 2.4 panel design is based on that of version 2.0, but contained five rams not present on version 2.0, and 78 rams that were not present on version 1.1. The 96 rams of version 2.4 sired twin offspring with known ewes, and are thus part of the 384-member USMARC Sheep Diversity Family Panel version 2.45.
Efficient and accurate codon scoring is challenging when multiple adjacent SNPs are present in the codon. For example, the International Union of Biochemistry (IUB) ambiguity codes for the nucleotide consensus sequence for ovine PRNP codon 171 are "MRK", which represents these known codons at position 171: CAG (Glu), CGG (Arg), CAT (His), and AAG (Lys). One solution to this problem is to employ primer extension chemistry whereby an oligonucleotide primer binds to an adjacent sequence on each strand and synthesis DNA polymerase is used to extend the primer across one, two, or three SNPs with specific mixtures of deoxy- and dideoxynucleotides (dNTPs and ddNTPs). The advantage over chemistries that employ only ddNTPs and are designed to extend exactly one nucleotide, is the mass of extended oligonucleotides generated from dNTPs and ddNTPs provides information about the haplotype status of the alleles. When both DNA strands from both alleles are interrogated in the same reaction, their respective results must be consistent if they are to be believed. This provides a convenient control that is internal to the biochemical reaction. The oligonucleotides for ovine PRNP amplification and MALDI-TOF MS testing are provided in Additional File 2. 350c69d7ab