Dataset Information

CCPRD: A Novel Analytical Framework for the Comprehensive Proteomic Reference Database Construction of NonModel Organisms.

ABSTRACT: Protein reference databases are a critical part of producing efficient proteomic analyses. However, the method for constructing clean, efficient, and comprehensive protein reference databases of nonmodel organisms is lacking. Existing methods either do not have contamination control procedures, or these methods rely on a three-frame and/or six-frame translation that sharply increases the search space and the need for computational resources. Herein, we propose a framework for constructing a customized comprehensive proteomic reference database (CCPRD) from draft genomes and deep sequencing transcriptomes. Its effectiveness is demonstrated by incorporating the proteomes of nematocysts from endoparasitic cnidarian: myxozoans. By applying customized contamination removal procedures, contaminations in omic data were successfully identified and removed. This is an effective method that does not result in overdecontamination. This can be shown by comparing the CCPRD MS results with an artificially contaminated database and another database with removed contaminations in genomes and transcriptomes added back. CCPRD outperformed traditional frame-based methods by identifying 35.2-50.7% more peptides and 35.8-43.8% more proteins, with a maximum of 84.6% in size reduction. A BUSCO analysis showed that the CCPRD maintained a relatively high level of completeness compared to traditional methods. These results confirm the superiority of the CCPRD over existing methods in peptide and protein identification numbers, database size, and completeness. By providing a general framework for generating the reference database, the CCPRD, which does not need a high-quality genome, can potentially be applied to nonmodel organisms and significantly contribute to proteomic research.

SUBMITTER: Guo Q

PROVIDER: S-EPMC7331046 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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CCPRD: A Novel Analytical Framework for the Comprehensive Proteomic Reference Database Construction of NonModel Organisms.

Guo Qingxiang Q Li Dan D Zhai Yanhua Y Gu Zemao Z

ACS omega 20200617 25

Protein reference databases are a critical part of producing efficient proteomic analyses. However, the method for constructing clean, efficient, and comprehensive protein reference databases of nonmodel organisms is lacking. Existing methods either do not have contamination control procedures, or these methods rely on a three-frame and/or six-frame translation that sharply increases the search space and the need for computational resources. Herein, we propose a framework for constructing a cust ...[more]

PMID: 32637811

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Project description:BackgroundClean rooms of the Space Assembly Facility (SAF) at the Jet Propulsion Laboratory (JPL) at NASA are the final step of spacecraft cleaning and assembly before launching into space. Clean rooms have stringent methods of air-filtration and cleaning to minimize microbial contamination for exoplanetary research and minimize the risk of human pathogens, but they are not sterile. Clean rooms make a selective environment for microorganisms that tolerate such cleaning methods. Previous studies have attempted to characterize the microbial cargo through sequencing and culture-dependent protocols. However, there is not a standardized metagenomic workflow nor analysis pipeline for spaceflight hardware cleanroom samples to identify microbial contamination. Additionally, current identification methods fail to characterize and profile the risk of low-abundance microorganisms.ResultsA comprehensive metagenomic framework to characterize microorganisms relevant for planetary protection in multiple cleanroom classifications (from ISO-5 to ISO-8.5) and sample types (surface, filters, and debris collected via vacuum devices) was developed. Fifty-one metagenomic samples from SAF clean rooms were sequenced and analyzed to identify microbes that could potentially survive spaceflight based on their microbial features and whether the microbes expressed any metabolic activity or growth. Additionally, an auxiliary testing was performed to determine the repeatability of our techniques and validate our analyses. We find evidence that JPL clean rooms carry microbes with attributes that may be problematic in space missions for their documented ability to withstand extreme conditions, such as psychrophilia and ability to form biofilms, spore-forming capacity, radiation resistance, and desiccation resistance. Samples from ISO-5 standard had lower microbial diversity than those conforming to ISO-6 or higher filters but still carried a measurable microbial load.ConclusionsAlthough the extensive cleaning processes limit the number of microbes capable of withstanding clean room condition, it is important to quantify thresholds and detect organisms that can inform ongoing Planetary Protection goals, provide a biological baseline for assembly facilities, and guide future mission planning. Video Abstract.

Project description:BackgroundSeveral recent studies have demonstrated the use of Roche 454 sequencing technology for de novo transcriptome analysis. Low error rates and high coverage also allow for effective SNP discovery and genetic diversity estimates. However, genetically diverse datasets, such as those sourced from natural populations, pose challenges for assembly programs and subsequent analysis. Further, estimating the effectiveness of transcript discovery using Roche 454 transcriptome data is still a difficult task.ResultsUsing the Roche 454 FLX Titanium platform, we sequenced and assembled larval transcriptomes for two butterfly species: the Propertius duskywing, Erynnis propertius (Lepidoptera: Hesperiidae) and the Anise swallowtail, Papilio zelicaon (Lepidoptera: Papilionidae). The Expressed Sequence Tags (ESTs) generated represent a diverse sample drawn from multiple populations, developmental stages, and stress treatments. Despite this diversity, > 95% of the ESTs assembled into long (> 714 bp on average) and highly covered (> 9.6x on average) contigs. To estimate the effectiveness of transcript discovery, we compared the number of bases in the hit region of unigenes (contigs and singletons) to the length of the best match silkworm (Bombyx mori) protein--this "ortholog hit ratio" gives a close estimate on the amount of the transcript discovered relative to a model lepidopteran genome. For each species, we tested two assembly programs and two parameter sets; although CAP3 is commonly used for such data, the assemblies produced by Celera Assembler with modified parameters were chosen over those produced by CAP3 based on contig and singleton counts as well as ortholog hit ratio analysis. In the final assemblies, 1,413 E. propertius and 1,940 P. zelicaon unigenes had a ratio > 0.8; 2,866 E. propertius and 4,015 P. zelicaon unigenes had a ratio > 0.5.ConclusionsUltimately, these assemblies and SNP data will be used to generate microarrays for ecoinformatics examining climate change tolerance of different natural populations. These studies will benefit from high quality assemblies with few singletons (less than 26% of bases for each assembled transcriptome are present in unassembled singleton ESTs) and effective transcript discovery (over 6,500 of our putative orthologs cover at least 50% of the corresponding model silkworm gene).

Dataset Information

CCPRD: A Novel Analytical Framework for the Comprehensive Proteomic Reference Database Construction of NonModel Organisms.

Publications

CCPRD: A Novel Analytical Framework for the Comprehensive Proteomic Reference Database Construction of NonModel Organisms.

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