Advanced Plant EXperiments-02-2 (APEX-02-2) - 04.26.17

Overview | Description | Applications | Operations | Results | Publications | Imagery

ISS Science for Everyone

Science Objectives for Everyone
For the Advanced Plant EXperiments-02-2 (APEX-02-2) investigation, a genome-wide series of deletion clones of Saccharomyces cerevisiae, or “Baker’s yeast”, is assayed for radiation damage during spaceflight in comparison to ground controls. On return to Earth, the yeast arrays from space and ground are analyzed by state-of-the-art next generation DNA sequencing techniques. This fresh application of DNA sequencing aims to determine the molecular mechanisms of radiation damage in order to facilitate understanding radiation damage, and may provide simple approaches to enhancing space based and clinical radiation damage.
Science Results for Everyone
Information Pending

The following content was provided by Timothy G. Hammond, M.B.B.S., and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom: APEX-02

Principal Investigator(s)
Timothy G. Hammond, M.B.B.S., Durham Veterans' Affairs Medical Center, Durham, NC, United States

Co-Investigator(s)/Collaborator(s)
Corey Nislow, seqWell Inc., Beverly, MA, United States
Holly H. Birdsall, M.D., Ph.D., Department of Veterans Affairs Office of Research and Development, Washington, DC, United States

Developer(s)
NASA Kennedy Space Center, Cape Canaveral, FL, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
NASA Research Office - Space Life and Physical Sciences (NASA Research-SLPS)

Research Benefits
Space Exploration, Earth Benefits, Scientific Discovery

ISS Expedition Duration
April 2017 - September 2017

Expeditions Assigned
51/52

Previous Missions
APEX-02 builds 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.

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Experiment Description

Research Overview

  • The research team proposes 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 undergoes 20 generations of growth, equivalent to approximately 400 human reproductive generations.
  • To properly survey the effects of radiation on this model organism, the research team uses whole genome sequencing of 1000 genomes. This cohort comprises of 100 individual strains, 90 of which contain verified deletion alleles in a variety of conserved pathways. Each 5 individual, independent clones of each strain are 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 are sequenced in parallel.
  • In previous experience, 100 unique deletion alleles 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 the Advanced Plant EXperiments-02-2 (APEX-02-2) investigation builds upon previous data to better understand the mechanisms of radiation damage, and may provide simple approaches to improving space based and clinical radiation damage.
  • The data sets are to be placed on a publicly assessable website as a community resource.

Description

Beginning in 2005, developments in massively parallel sequencing Next Generation Sequencing (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. Epigenomics, 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, 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 spaceflight mission. A genome-wide series of deletion clones are to be assayed to study the genomics of radiation effects. The research team proposes to return the yeast arrays from space, analyze the DNA by next generation sequencing, and make comparison to ground controls. Pathway analysis then allows analysis of known and novel radiation sensitive pathways. The data sets are to be placed on a publicly assessable website as a community resource.
 
The research team uses high-throughput, NGS to assess the effects of long term exposure to radiation using the yeast, Saccharomyces cerevisiae. Although the chronological time on station is limited, each strain undergoes 20 generations of growth, equivalent to approximately 400 human reproductive generations. To properly survey the effects of radiation on this model organism, The research team uses whole genome sequencing of 1000 genomes. This cohort comprises of 100 individual strains, 90 of which contain verified deletion alleles in a variety of conserved pathways. Each 5 individual, independent clones of each strain are 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 are sequenced in parallel. In previous experience, 100 unique deletion alleles provide a robust data set for reconstructing pathways that are both directly and indirectly involved in the cells resistance to radiation damage.
 
The research team has over 10 years of experience using collections of yeast mutants for dissecting the DNA damage response, and more recently, 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 is extracted and sequenced according to published protocol (Hill et al., PLoS Genet. 2013 Apr;9(4):e1003390).
 
Yeast genomes are 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 are 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 are 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 are 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, and 3) is multi-threaded with a minimal memory. Alignments and all subsequent sequence data are 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 are 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 International Space Station (ISS) vs. ground controls. All variant positions require a minimum coverage of 15X to be considered as a candidate SNV.
 
The software package CNV-seq is used to identify chromosomal regions that varied in copy number between parental strains and experimental samples. Sequence data is to be made publicly available on the NCBI Short Read Archive and NASA’s GeneLab Data Archives.

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Applications

Space Applications
The APEX-02-2 work quantitatively measures 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.

Earth Applications
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 can 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 builds upon previous data to understanding the mechanisms of radiation damage, and may provide simple approaches to mitigating clinical radiation damage.

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Operations

Operational Requirements and Protocols
The 33 petri plates are launched in Cold Stowage at +4°C. The plates are then transferred to ambient stowage on ISS. This activates yeast growth. The plates are then returned to Earth at ambient on same vehicle as ascent.

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Decadal Survey Recommendations

Information Pending

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Results/More Information

Information Pending

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Related Websites

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Imagery