Assessment of REPLI-g Multiple Displacement Whole Genome Amplification (WGA) Techniques for Metagenomic Applications
Year: 2017
Type: article
Abstract: Amplification of minute quantities of DNA is a fundamental challenge in low-biomass metagenomic and microbiome studies because of potential biases in coverage, guanine-cytosine (GC) content, and altered species abundances.Whole genome amplification (WGA), although widely used, is notorious for introducing artifact sequences, either by amplifying laboratory contaminants or by nonrandom amplification of a sample's DNA.In this study, we investigate the effect of REPLI-g multiple displacement amplification (MDA; Qiagen, Valencia, CA, USA) on sequencing data quality and species abundance detection in 8 paired metagenomic samples and 1 titrated, mixed control sample.We extracted and sequenced genomic DNA (gDNA) from 8 environmental samples and compared the quality of the sequencing data for the MDA and their corresponding non-MDA samples.The degree of REPLI-g MDA bias was evaluated by sequence metrics, species composition, and crossvalidating observed species abundance and species diversity estimates using the One Codex and MetaPhlAn taxonomic classification tools.Here, we provide evidence of the overall efficacy of REPLI-g MDA on retaining sequencing data quality and species abundance measurements while providing increased yields of high-fidelity DNA.We find that species abundance estimates are largely consistent across samples, even with REPLI-g amplification, as demonstrated by the Spearman's rank order coefficient (R 2 .0.8).However, REPLI-g MDA often produced fewer classified reads at the species, genera, and family level, resulting in decreased species diversity.We also observed some areas with the PCR "jackpot effect," with varying input DNA values for the Metagenomics Research Group (MGRG) controls at specific genomic loci.We visualize this effect in whole genome coverage plots and with sequence composition analyses and note these caveats of the MDA method.Despite overall concordance of species abundance between the amplified and unamplified samples, these results demonstrate that amplification of DNA using the REPLI-g method has some limitations.These concerns could be addressed by future improvements in the enzymes or methods for REPLI-g to be considered a .99%robust method for increasing the amount of high-fidelity DNA from low-biomass samples or at the very least, accounted for during computational analysis of MDA samples.
Institutions: Weill Cornell Medicine, New York Medical College
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
Funders: National Aeronautics and Space Administration, Bill and Melinda Gates Foundation, Alfred P. Sloan Foundation, Vallee Foundation, Pershing Square Sohn Cancer Research Alliance, WorldQuant Foundation, National Institutes of Health, Weill Cornell Medical College, National Institute of Allergy and Infectious Diseases, National Institute of Neurological Disorders and Stroke, National Institute of Environmental Health Sciences, National Institute of Biomedical Imaging and Bioengineering
Awards: G1284901464, (1) SUMMARYMASSIVELY PARALLEL DNA SEQUENCING CONTINUES TO PROVIDE ASTONISHING INSIGHTS INTO THE REGULATORY COMPLEXITY OF THE MAMMALIAN GENOME REVEALING HERITABLE AND DE NOVO OCCURRING RISK FACTORS PREDISPOSING HUMANS TO A WIDE VARIETY OF DISEASES AND DISORDERS. NOTABLY MEMBERS OF THE ENCYCLOPEDIA OF DNA ELEMENTS (ENCODE) CONSORTIUM INCLUDING CO-INVESTIGATORS DRS. MASON AND SNYDER HAVE RECENTLY MAPPED THOUSANDS OF NON-CODING REGULATORY ELEMENTS IN THE HUMAN GENOME THAT SPECIFY RISK AND PROTECTIVE SITES FOR MANY DISEASE. THESE NONCODING GENETIC VARIATIONS ALSO SHOW DIFFERENTIAL IMPACT DEPENDING ON THE EPIGENETIC CHANGES OF THE GENOME INCLUDING METHYLATION OF DNA AND CHROMATIN STATES INSIDE CELLS. YET THESE QUESTIONS HAVE NOT BEEN COMPREHENSIVELY EXAMINED IN HUMAN ASTRONAUTS DUE TO THE FACT THAT THE VERY FIRST DATA SETS THAT CAN DELINEATE THE PUTATIVE IMPACT OF GENOME-WIDE NONCODING VARIATION AND EPIGENETIC CHANGES FROM SPACE TRAVEL ARE ONLY NOW BEING GENERATED IN THE NASA TWINS STUDY.HOWEVER WITH THIS DATA FROM NASA TWINS STUDY THERE IS A UNIQUE OPPORTUNITY TO BOTH GENERATE NEW COMPUTATIONAL TOOLS FOR THE INTEGRATION OF THE DATA FROM THESE SAMPLES AND ALSO TO UTILIZE NOVEL SINGLE-CELL APPROACHES FOR EPIGENETIC STATES IN THE HUMAN CELLS TAKEN FROM SPACE. WE PROPOSE TO EXPAND AND FURTHER INTEGRATE OUR TOOLS FOR EXAMINING THE REGULATORY CHANGES TO DNA AND RNA TOGETHER CALLED "EPISUITE " WHICH WILL PROVIDE LONG-SOUGHT SOLUTIONS TO PROBLEMS IN INTEGRATIVE GENOMICS AND ESTABLISH A MORE SYSTEMS-LEVEL VIEW OF HUMAN PHYSIOLOGY DURING SPACE TRAVEL. SPECIFICALLY WE WILL OPTIMIZE AND INTEGRATE OPEN-SOURCE AND PUBLISHED BIOINFORMATICS TOOLS BETWEEN BOTH LABORATORIES (HUGE-SEQ METHYLKIT EDMR R-MAKE) EXPAND OUR CODE BASE WITH TWO NEW TOOLS (METHCLONE AND CEMBASE) AND THEN CREATE A WEB-INTERFACE FOR USERS TO UPLOAD ORGANIZE AND ANALYZE THESE DATA RELATIVE TO ENCODE PUBLIC DATA. THEN WE WILL USE NOVEL SINGLE-CELL EXPERIMENTAL METHODS (SCATAC-SEQ) TO CONFIRM AND CHARACTERIZE THE IMPACT AND SCOPE OF SUCH NONCODING AND EPIGENETIC CHANGES. TOGETHER THE DATA AND TOOLS IN EPISUITE CONCOMITANT WITH THE NEW SINGLE-CELL CHROMATIN DATA WILL ENABLE DISCOVERY AND VALIDATION OF SPECIFIC LOCI THAT REPRESENT THE GENETIC AND EPIGENETIC RISKS FACED BY ASTRONAUTS AND ALSO AN IMPROVED UNDERSTANDING OF THE REGULATORY AND GENETIC IMPACT (BMED5) OF THESE MOLECULAR CHANGES.(2) HYPOTHESISWE HYPOTHESIZE THAT SPECIFIC AREAS OF THE GENOME ARE EPIGENETICALLY DYSREGULATED AS A RESULT OF SPACE TRAVEL AND THAT SOME OF THESE AREAS DIFFER BETWEEN ASTRONAUTS. MOREOVER WE HYPOTHESIZE THAT THE ENRICHMENT OF NON-CODING VARIANTS IN DISTINCT BIOLOGICAL PATHWAYS WILL ALSO DIFFER BETWEEN ASTRONAUTS AND ANALYSIS AND INTEGRATION OF THESE DATA WILL REVEAL THE FIRST REGIONS OF "EPIGENETIC SPACE RISK" FOR THE ASTRONAUTS DELINEATED IN BMED5. TO VALIDATE THESE PREDICTIONS WE WILL PERFORM SINGLE-CELL ATAC-SEQ (ASSAY FOR TRANSPOSASE ACCESSIBLE CHROMATIN) ON CELL FRACTIONS FROM EARTH-BASED CONTROL SAMPLES AND THOSE TAKEN FROM SPACE.(3) SPECIFIC AIMSAIM 1. CREATE A WEB-BASED FRAMEWORK FOR INTEGRATING NON-CODING GENOMIC VARIATION AND DIFFERENTIAL EPIGENETIC STATES. THIS EPISUITE INTERFACE AND SET OF MACHINE-LEARNING ALGORITHMS WILL HELP ONGOING WORK WITH THE NASA TWINS STUDY AND AID FUTURE INVESTIGATIONS THAT LEVERAGE MULTI-OMICS DATA SETS FROM ASTRONAUTS. THESE DATA WILL ALSO BE LINKED TO GENE EXPRESSION DATA TO GAUGE THE IMPACT OF GENETIC AND EPIGENETIC CHANGES ON SPECIFIC BIOLOGICAL NETWORKS.AIM 2. VALIDATE THE ALTERED CHROMATIN STATES IN CELLS FROM SPACE TRAVEL AND MEASURE THE PROPORTION OF CELLS CHANGED WITH SINGLE-CELL ATAC-SEQ. WE WILL USE AN ASSAY CALLED SINGLE CELL (SCATAC-SEQ) WHICH CAN MEASURE THE SITES OF OPEN AND CLOSED CHROMATIN ON A CELL-BY-CELL BASIS. THIS WILL VALIDATE THE SITES WHERE WE OBSERVE EPIGENETIC CHANGES AND ALSO PROVIDE THE FIRST ESTIMATE OF THE PROPORTION OF CELLS EPIGENETICALLY IMPACTED BY SPACE TRAVEL., G232304109, NEXT-GENERATION SEQUENCING (NGS) TECHNOLOGIES HAVE RECENTLY BEEN USED TO DETECT AND CLASSIFY EPIGENETIC CHANGES AT AN UNPRECEDENTED LEVEL. EPIGENETIC, G4198848887, Community Research Education and Engagement for Data Science (CREEDS), Study of Neural Tube Defects Etiology: Genome and Exposome, Joan & Sanford I. Weill Medical College of Cornell University - to monitor the spread of antimicrobial resistance by generating a global map of bacterial species, resistance genes and markers, and modified bases..., G6978705394, Research - Weill Cornell Medical College (2015), G7535378661, Epigenome Interactions in Complex Neurogenetic Disorders, G9000163900
OpenAlex ID: W2602820708