Project description:Purpose: This study uses a high-throughput glycan microarray to develop a novel method to assign ABO blood type. The method will then be applied to samples from patients treated with PROSTVAC to determine if blood type correlates with survival Results: Many blood group A and B antigens correlate with blood type. Blood typing is best achieved using a combination of 10 signals Conclusion: ABO blood type can be determined with greater than 97% accuracy using only 4 microliters of serum.
Project description:Chagas disease is one of the most important neglected diseases with an estimated number of 12 million infected individuals, the majority living in Central and South America. The Trypanosoma cruzi (T.cruzi) protozoan parasite is the etiological agent of Chagas disease. T.cruzi is highly genetically diverse and a new nomenclature assigned each strain to seven genetic groups (TcI-TcVI and Tcbat), named Discrete Typing Units (DTUs), based on their biochemical, immunological and phenotypical characteristics. T.cruzi DTUs have been correlated to diverse clinical outcomes highlighting the importance of molecular epidemiological screens. Despite the development of T.cruzi typing methods based on genetic signatures, each method presenting its own advantages and challenges. The work presented here shows the application of mass spectrometry for Trypanosoma cruzi Strain Typing Assay using MS2 peptide spectral libraries (Tc-STAMS2). The novelty of the method is based on the use of peptide fragmentation spectra as strain-specific fingerprints to classify and identify DTUs. Initially, a spectra library is generated from characterized T.cruzi strains. The library is subsequently inspected using MS/MS spectra from unknown strains and confidently assigned to a specific strain in an automated and computationally-driven approach. The Tc-STAMS2 method was challenged to test several variables such as sample type and preparation, instrument setup and identification platform. Tc-STAMS2 provided high confidence and robustness in T.cruzi strain typing. The Tc-STAMS2 method represents a proof-of-concept of a complementary strategy to the current DNA-based T. cruzi genotyping methods. Moreover, the method allows the identification of strain-specific features that could be related to the biology of T.cruzi strains and their clinical outcomes.
Project description:Comparison of High Resolution Viruelnce Allelic Profiling (HReVAP) typing with Multilocus Sequence Typing and Whole Genome SNPs analysis for typing VTEC strains
Project description:Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), were validate with Illumina HumanHap550-Duo Genotyping Beadchip. As seen in (Suppl. Table 5a), the call rates (97.30% to 99.07%) and accuracy or concordance ( > 99.85% for the SNPs called in both amplicon and natural reference) for 5 ng derived amplicons with both Wpa and Gv2 were close to each other and close to native gDNA (call rate: 98.3% to 99.75%). These call rates were better than a recent report (amplicon 95.9% vs. un-amplified 98.5%), in which the early kit Repli-g 625S was applied, and re-genotyping was performed when the performance was low and duplicate samples were filtered for the highest call rate. The genotyping accuracy of Wpa was actually in the same range as the variation in technical replicates with similar SNP typing arrays (99.87% and 99.88%, replicated Affymetrix array, or between Affymetrix and Illumina arrays). Importantly, the genotyping concordance for amplicons generated from 0.5 ng with Wpa (99.88% and 99.69%) were also close to the technical replicates. In this case, the call rates of Wpa were slightlyreduced compared to that with 5 ng input, but the call rate for the partially degraded sample 04-018, was modestly improved over Gv2 (92.06 % vs. 90.53%). Wpa data also showed some amplification non-uniformity among different locations, resulting in some “artificial CNVs” similar to Gv2 (exampled as in Suppl. Fig. 5 and Suppl. Table 6), with the outputs obtained by taking unamplified gDNAs as their reference. This imbalance however was consistent and reproducible for each method but different between Wpa and Gv2. These artificial CNVs can be efficiently cancelled if pair-wise amplified test and reference are compared, as observed in CGH result (Fig. 4 and Suppl. Fig. 4), also supported by others {Pugh 2008}. It is interesting to note that the representation of chromosomal terminal sequences was greatly improved with Wpa compared with Gv2 (Fig. 5), and that some of these regions were significantly under-amplified or even lost with Gv2 (Suppl. Fig. 5 and Suppl. Table 6, 7), as also independently reported recently {Pugh 2008}. This occurred especially in the terminal 3 to 5 Mb and sometimes extended to 10 Mb in many chromosome termini, and was particularly serious when low levels or degraded DNA was as input. An analysis for 5 Mb termini is shown (Suppl. Table 5b calculated all involved SNPs as a cohort. Fig. 5 and Suppl. Tables 6 and 7 were the result for each chromosome terminus). Importantly, the SNP typing was also greatly improved, outstandingly exemplified by the amplicons of 0.5 ng input for the partially degraded 04-018, with Wpa versus Gv2 call rate of 91.9% vs. 84.45% and accuracy of 99.57% vs. 98.62%. The result also showed that these terminal regions underrepresentation in Gv2 was not absolutely associated with the distance-to-end, but possibly was a sequence related issue. Keywords: Whole-pool amplification, whole genome SNP typing