Advanced Plant EXperiments-02-2 (APEX-02-2) - 07.14.16
A genome-wide series of deletion clones of Saccharomyces cerevisiae or “Baker’s yeast” was assayed for radiation damage during spaceflight in comparison to ground controls. The yeast colonies flown in space are invariably exposed to space based radiation. On return the yeast arrays from space and ground were analyzed by state-of-the-art next generation DNA sequencing techniques. Pathway analysis identified novel radiation sensitive pathways. To translate this data to clinical and space health countermeasures, 10 new deletion series have been created with a second putative control genes also deleted. This fresh application aims to determine the molecular mechanisms of radiation damage to facilitate understanding radiation damage, and may provide simple approaches to ameliorating space based and clinical radiation damage. The data sets will be placed on a publicly assessable website as a community resource. Science Results for Everyone
Information Pending Experiment Details
Timothy G. Hammond, M.B.B.S., Durham Veterans' Affairs Medical Center, Durham, NC, United States
Holly H. Birdsall, M.D., Ph.D., Department of Veterans Affairs Office of Research and Development, Washington, DC, United States
Corey Nislow, seqWell Inc., Beverly, MA, United States
NASA Kennedy Space Center, Cape Canaveral, FL, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
NASA Research Office - Space Life and Physical Sciences (NASA Research-SLPS)
Space Exploration, Earth Benefits, Scientific Discovery
ISS Expedition Duration
September 2016 - March 2017
APEX-02 will build upon the previously flown experiments which flew on STS-86, STS-89, STS-90, STS-106, STS-105, STS-108, STS-112, Expedition 8 and 9, STS-115, STS-135, and the National Lab Pathfinder missions on STS-123, STS-124, STS-119, STS-125, STS-126, STS-128, STS-129, STS-130, STS-131, STS-132, STS-133, STS-134, SpaceX-3, SpaceX-4, SpaceX-7, and Space X-8.
• We propose to use high-throughput, next-generation sequencing (NGS) to assess the effects of long term exposure to radiation using the yeast, Saccharomyces cerevisiae. Each strain will undergo 20 generations of growth, equivalent to approximately 400 human reproductive generations. • To properly survey the effects of radiation on this model organism, we will use whole genome sequencing of 1000 genomes. This cohort will comprise 100 individual strains, 90 of which contain verified deletion alleles in a variety of conserved pathways. Each 5 individual, independent clones of each strain will be sequenced, along with 5 isolates of 10 well-characterized controls strains that are deleted for known components of the DNA damage and repair response Identical numbers of ground controls will be sequenced in parallel. • In our experience, 100 unique deletion alleles will provide a robust dataset for reconstructing pathways that are both directly and indirectly involved in the cells resistance to radiation damage. • The hope is that knowledge gained from this investigation will build upon previous data to understanding the mechanisms of radiation damage, and may provide simple approaches to ameliorating space based and clinical radiation damage. • The data sets will be placed on a publicly assessable website as a community resource.
Beginning in 2005, developments in massively parallel sequencing Next Generation Sequencing or NGS changed the way in which DNA sequencing was performed. This revolutionized the field of genomics by delivering a complete human genome sequences in a fraction of the time (days versus years) and at a fraction of the cost (thousands of dollars versus billions of dollars). No aspect of life sciences has been untouched by this revolution and new fields have emerged, such as ecological genomics, microbiome genomics and personalized genomics. The microbiome (all the microbes present in diverse body sites) is becoming a key diagnostic of the health of the host. Epi-genomics, the study of DNA modifications, including those that promote serious diseases, is now possible, as are the delineation of complex traits that can determine susceptibility to disease and offer insights on treatment. The recent Decadal Survey Report, “Recapturing a Future for Space Exploration: Life and Physical Sciences Research for a New Era,” by the National Research Council (http://www.nap.edu/catalog.php?record_id=13048), specifically mentions the need for NASA to utilize new Omics research technologies. The biologic effects of radiation in space continue to pose a challenge to space exploration and Omics may provide useful new tools to apply to this problem. The current proposal is to fly a yeast payload on an upcoming space flight mission. A genome-wide series of deletion clones will be assayed to study the genomics of radiation effects. We propose to return the yeast arrays from space and analyze the DNA by next generation sequencing and make comparison to ground controls. Pathway analysis will then allow analysis of known and novel radiation sensitive pathways. The data sets will be placed on a publicly assessable website as a community resource. We propose to use high-throughput, next-generation sequencing (NGS) to assess the effects of long term exposure to radiation using the yeast, Saccharomyces cerevisiae. Although the chronological time on station will be limited, each strain will undergo 20 generations of growth, equivalent to approximately 400 human reproductive generations. To properly survey the effects of radiation on this model organism, we will use whole genome sequencing of 1000 genomes. This cohort will comprise 100 individual strains, 90 of which contain verified deletion alleles in a variety of conserved pathways. Each 5 individual, independent clones of each strain will be sequenced, along with 5 isolates of 10 well-characterized controls strains that are deleted for known components of the DNA damage and repair response (see table below). Identical numbers of ground controls will be sequenced in parallel. In our experience, 100 unique deletion alleles will provide a robust dataset for reconstructing pathways that are both directly and indirectly involved in the cells resistance to radiation damage. We have over 10 years of experience using collections of yeast mutants for dissecting the DNA damage response, and more recently, we have pioneered the use of next-generation sequencing to rapidly and cost-effectively decode complete yeast genomes and identify novel mutations that arise in response to a variety of perturbations. Control “hypermutable” Strains ORF GENE Description YBR136W MEC1 Genome integrity checkpoint protein and PI kinase superfamily member YCR066W RAD18 E3 ubiquitin ligase YER095W RAD51 Strand exchange protein YER162C RAD4 Protein that recognizes and binds damaged DNA (with Rad23p) during NER YER173W RAD24 Checkpoint protein YJR035W RAD26 Protein involved in transcription-coupled nucleotide excision repair YJR052W RAD7 Protein that binds damaged DNA during NER YKL113C RAD27 5' to 3' exonuclease, 5' flap endonuclease YLR032W RAD5 DNA helicase YMR224C MRE11 Nuclease subunit of the MRX complex with Rad50p and Xrs2p NGS protocol Yeast DNA will be extracted and sequenced according to our published protocol (Hill et al., PLoS Genet. 2013 Apr;9(4):e1003390). Yeast genomes will be sequenced in a multiplexed format, where an oligonucleotide index barcode was embedded within adapter sequences ligated to genomic DNA fragments. Only one mismatch per barcode is permitted to prevent contamination across samples and sequence reads will be filtered for low quality base calls trimming all bases from 5′ and 3′ read ends with Phred scores < Q30. Trimming sequence reads for low quality base calls to lower false positive SNV calls. De-multiplexed and trimmed reads from the S. cerevisiae strains will be aligned to the S288c 2010 genome, a high fidelity sequence from an individual yeast colony (from F. Dietrich's lab at Duke University; it is the SGD reference genome as of February 2011). Sequence reads will be aligned with Bowtie2, one of the fastest, and most accurate aligner that is 1) updated frequently, 2) supports variable read lengths within a single input file, 3)I s multi-threaded with a minimal memory. Alignments and all subsequent sequence data will be visualized using the Savant Genome Browser. The average coverage of each genome will be 50X to ensure confident variant detection. Aligned sequence reads for S. cerevisiae will be processed using the Unified Genotyper package of the Genome Analysis Toolkit (GATK), which features a comprehensive framework for discovering SNVs and calculating coverage across genomic data. Variants detected in the S. cerevisiae parental strains will be subtracted from complete variant lists, yielding a set of novel variants that emerged during strain growth during growth on the ISS vs. ground controls. All variant positions will require a minimum coverage of 15× to be considered as a candidate SNV. The software package CNV-seq will be used to identify chromosomal regions that varied in copy number between parental strains and experimental samples. Sequence data will be made publicly available on the NCBI Short Read Archive http://www.ncbi.nlm.nih.gov/sra and NASA’s GeneLab Data Archives.
The APEX-02-2 work will quantitatively measure radiation damage to yeast DNA exposed to space radiation.. Using state of the art technologies, the science team can, for the first time, conduct a highly powered, genome wide analysis of mechanisms of radiation damage in space.. Knowledge gained from this investigation will build upon previous data to understanding the mechanisms of radiation damage, and may provide simple approaches to ameliorating space based radiation damage.
By using yeast strains to understand the effects of space based radiation stresses at the cellular level, a greater knowledge of the regulatory mechanisms at work in cells will be gained. A hallmark of radiation stresses is that they induce physiological responses; primary among them are DNA damage. As yeast is a model eukaryotic organism, DNA damage changes caused by radiation could lead to a greater understanding of these processes on earth, thus benefiting all citizens. Knowledge gained from this investigation will build upon previous data to understanding the mechanisms of radiation damage, and may provide simple approaches to ameliorating clinical radiation damage.
Operational Requirements and Protocols
The 33 petri plates will be launched in Cold Stowage at +4 C. The plates will then be transferred to ambient stowage on ISS. This will activate yeast growth. The plates will then return at ambient on same vehicle as ascent.
Decadal Survey Recommendations
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