The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3–12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled “Polymerase-exonuclease (PEX) PCR”, in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3’ end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.
Draft genome sequences of oyster-associated Pseudomonas stutzeri strain MF28, P. alcaligenes strain OT69, P. aeruginosa strain WC55, Stenotrophomonas maltophilia strain MF89, and Microbacterium maritypicum strain MF109 are reported. Genome-wide surveys of these isolates suggest that the oyster microbiome, which remains largely understudied, has a strong potential to degrade crude oil.
Genome sequences for three strains of denitrifying bacteria (Alphaproteobacteria—Afipia sp. strain 1NLS2 and Hyphomicrobium denitrificans strain 1NES1; Firmicutes—Bacillus sp. strain 1NLA3E) isolated from the nitrate- and uranium-contaminated subsurface of the Oak Ridge Integrated Field Research Challenge (ORIFRC) site, Oak Ridge Reservation, TN, are reported.
Colorectal cancer (CRC) is one of the major causes of global mortality. Recent clinical advances in T cell therapy using antibodies that block inhibitory T cell signals have been transforming cancer therapy. Studies show that somatic mutations in protein coding regions produce tumor-specific antigens, or neoantigens. CRC warrants deeper investigation into the non-synonymous exonic mutations that seem to be drivers in tumor development. Tumor mutation burden (TMB) and the abundance of neoantigens are highly correlated and it has been shown that TMB can be estimated using targeted sequencing panels. The aim of this study is to investigate 10 matched tumor/normal CRC samples using a panel targeting 422 genes covering about 1.7 Mb to screen for potential neoepitope candidates. Although sequencing and bioinformatics pipelines are developed to effectively filter neoantigens, it is still unclear which antigens elicit a T cell response. A review of the tools used in neoepitope prediction shows that it is not only a very complex task, but demands high sequencing depth. Comparing currently available tools will enable us to understand the strengths and shortcomings of each algorithm, and develop a custom pipeline framework.
Note: This abstract was not presented at the meeting.
Citation Format: Raghavee Venkatramanan, Kerry Deutsch, Inah Golez, Pallavi Shroff, Spencer Seale, Brendan Ralph, Sally. Dow, Evan Anderson, Saman Tahir, Anjali Malge, Corey Braastad, Steven Anderson. Neoepitope screening in colorectal cancer patients by custom exome panel profiling somatic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1679.
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