``sage 10 - includes advances of sage

Upload: satyam-singh

Post on 03-Jun-2018

224 views

Category:

Documents


0 download

TRANSCRIPT

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    1/64

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    2/64

    SAGE TECHNOLOGY AND

    ITS APPLICATIONS

    PRESENTED BYDr. R.A.Siddique &

    Dr.Anand Kumar

    Animal Biochemistry Division

    N.D.R.I., Karnal (Haryana)India, 132001

    E-mail: [email protected]

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    3/64

    WHAT IS SAGE?

    Serial analysis of gene expression (SAGE) isa powerful tool that allows digital analysis ofoverall gene expression patterns.

    Produces a snapshot of the mRNA populationin the sample of interest.

    SAGE provides quantitative andcomprehensive expression profiling in a givencell population.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    4/64

    SAGE invented at Johns Hopkins University in

    USA (Oncology Center) by Dr. Victor Velculescuin 1995.

    An overview of a cellscomplete gene activity.

    Addresses specific issues such as determination ofnormal gene structure and identification of

    abnormal genome changes.

    Enables precise annotation of existing genes anddiscovery of new genes.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    5/64

    NEED FOR SAGE..

    Gene expression refers to the study of howspecific genes are transcribed at a given point intime in a given cell.

    Examining which transcripts are present in a cell.

    SAGE enables large scale studies of DNAexpression; these can be used to create'expression profiles.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    6/64

    Allows rapid, detailed analysis of thousands of

    transcripts in a cell.

    By comparing different types of cells, generate

    profiles that will help to understand healthy cellsand what goes wrong during diseases.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    7/64

    THREE PRINCIPLES UNDERLIE THE

    SAGE METHODOLOGY:

    A short sequence tag (10-14bp) contains sufficientinformation to uniquely identify a transcript provided that

    the tag is obtained from a unique position within eachtranscript

    Sequence tags can be linked together to from long serialmolecules that can be cloned and sequenced

    Quantitation of the number of times a particular tag isobserved provides the expression level of thecorresponding transcript.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    8/64

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    9/64

    PRE REQUISITES:

    Extensive sequencing techniques

    Deep bioinformatic knowledge

    Powerful computer software (assemble and analyze resultsfrom SAGE experiments)

    Limited use of this sensitive technique inacademic research laboratories

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    10/64

    STEPS IN BRIEF..

    1. Isolate the mRNA of an input sample (e.g. a

    tumour).

    2. Extract a small chunk of sequence from a

    defined position of each mRNA molecule.

    3. Link these small pieces of sequence together to

    form a long chain (or concatamer).

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    11/64

    4. Clone these chains into a vectorwhichcan be taken up by bacteria.

    5. Sequence these chains using modern high-throughput DNA sequencers.

    6. Process this data with a computer to countthe small sequence tags.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    12/64

    SAGE FLOWCHART

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    13/64

    SAGE TECHNIQUE (in detail)Trap RNAs with beads

    Messenger RNAs end with a long string of "As" (adenine)

    Adenine forms very strong chemical bonds with another nucleotide,thymine(T)

    Molecule that consists of 20 or so Ts acts like a chemical bait tocapture RNAs

    Researchers coat microscopic, magnetic beads with chemical baits i.e."TTTTT" tails hanging out

    When the contents of cells are washed past the beads, the RNAmolecules will be trapped

    A magnet is used to withdraw the bead and the RNAs out of the"soup"

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    14/64

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    15/64

    cDNA SYNTHESIS

    Double stranded cDNA is synthesized from the extracted

    mRNA by means of biotinylated oligo (dT) primer.

    cDNA synthesized is immobilised to streptavidin beads.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    16/64

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    17/64

    ENZYMATIC CLEAVAGE OF cDNA.

    The cDNA molecule is cleaved with a restrictionenzyme.

    Type II restriction enzyme used.

    Also known as Anchoring enzyme. E.g.NlaIII.

    Any 4 base recognising enzyme used.

    Average length of cDNA 256bp with sticky endscreated.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    18/64

    The biotinylated 3 cDNA are affinity purified using strepatavidin

    coated magnetic beads.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    19/64

    LIGATION OF LINKERS TO BOUND

    cDNA These captured cDNAs are divided into two

    halves, then ligated to linkers A and B,respectively at their ends.

    Linkers also known as docking modules.

    They are oligonucleotide duplexes.

    Linkers contain:

    NlaIII 4- nucleotide cohesive overhangType IIS recognition sequence

    PCR primer sequence (primer A or B).

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    20/64

    Type IIS restriction enzymetagging enzyme.

    Linker/docking module:

    PRIMER TE AE TAG

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    21/64

    CLEAVAGE WITH TAGGING

    ENZYME Tagging enzyme, usuallyBmsFIcleave DNA 14-

    15 nucleotides, releasing the linkeradapted

    SAGE tag from each cDNA.

    Repair of ends to make blunt ended tags using

    DNA polymerase (Klenow) and dNTPs.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    22/64

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    23/64

    FORMATION OF DITAGS What is left is a collection of short tags taken from each

    molecule.

    Two groups of cDNAs are ligated to each other, to create a

    ditag with linkers on either end.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    24/64

    Ligation using T4 DNA ligase.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    25/64

    PCR AMPLIFICATION OF

    DITAGS

    The linker-ditag-linker constructs are

    amplified by PCR using primers specific

    to the linkers.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    26/64

    ISOLATION OF DITAGS

    The cDNA is again digested by the AE.

    Breaking the linker off right where it was added in thebeginning.

    This leaves a sticky end with the sequence GTAC (orCATG on the other strand) at each end of the ditag.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    27/64

    CONCATAMERIZATION OF

    DITAGS Tags are combined into much longer molecules, called

    concatemers.

    Between each ditag is the AE site, allowing the scientistand the computer to recognize where one ends and the next

    begins.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    28/64

    CLONING CONCATAMERS

    AND SEQUENCING Lots of copies are required- So the concatemers are put

    into bacteria, which act like living "copy machines" to

    create millions of copies from the original

    These copies are then sequenced, using machines that canread the nucleotides in DNA. The result is a long list of

    nucleotides that has to be analyzed by computer

    Analysis will do several things: count the tags, determinewhich ones come from the same RNA molecule, and figureout which ones come from known, well-studied genes and

    which ones are new

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    29/64

    Quantitation of gene expression

    And data presentation

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    30/64

    How does SAGE work?1. Isolate mRNA.

    2.(b) Synthesize ds cDNA.

    2.(a) Add biotin-labeled dT primer:

    4.(a) Divide into two pools and add linker sequences:4.(b) Ligate.

    3.(c) Discard loose fragments.

    3.(a) Bind to streptavidin-coated beads.3.(b) Cleave with anchoring enzyme.

    5. Cleave with tagging enzyme.

    6. Combine pools and ligate.

    7. Amplify ditags, then cleave with anchoring enzyme.

    8. Ligate ditags.

    9. Sequence and record the tags and frequencies.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    31/64

    Vast amounts ofdata is produced, which

    must be sifted and ordered for useful

    information tobecome apparent.Sage reference databases:

    SAGE map

    SAGE Genie

    http://www.ncbi.nlm.nih.gov/cgap

    http://www.ncbi.nlm.nih.gov/cgaphttp://www.ncbi.nlm.nih.gov/cgap
  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    32/64

    What does the data look like?

    TAG COUNT TAG COUNT TAG COUNT

    CCCATCGTCC 1286 CACTACTCAC 245 TTCACTGTGA 150

    CCTCCAGCTA 715 ACTAACACCC 229 ACGCAGGGAG 142

    CTAAGACTTC 559 AGCCCTACAA 222 TGCTCCTACC 140

    GCCCAGGTCA 519 ACTTTTTCAA 217 CAAACCATCC 140

    CACCTAATTG 469 GCCGGGTGGG 207 CCCCCTGGAT 136

    CCTGTAATCC 448 GACATCAAGT 198 ATTGGAGTGC 136

    TTCATACACC 400 ATCGTGGCGG 193 GCAGGGCCTC 128

    ACATTGGGTG 377 GACCCAAGAT 190 CCGCTGCACT 127

    GTGAAACCCC 359 GTGAAACCCT 188 GGAAAACAGA 119

    CCACTGCACT 359 CTGGCCCTCG 186 TCACCGGTCA 118

    TGATTTCACT 358 GCTTTATTTG 185 GTGCACTGAG 118

    ACCCTTGGCC 344 CTAGCCTCAC 172 CCTCAGGATA 114

    ATTTGAGAAG 320 GCGAAACCCT 167 CTCATAAGGA 113

    GTGACCACGG 294 AAAACATTCT 161 ATCATGGGGA 110

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    33/64

    FROM TAGS TO GENES

    Collect sequence records from GenBank

    Assign sequence orientation (by finding poly-A

    tail or poly-A signal or from annotations)

    Extract 10-bases -adjacent to 3-most CATG Assign UniGene identifier to each sequence with a

    SAGE tag

    Record (for each tag-gene pair)

    #sequences with this tag

    #sequences in gene cluster with this tag

    Maps available at http://www.ncbi.nlm.nih.gov/SAGE

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    34/64

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    35/64

    DIFFERENTIAL GENE

    EXPRESSION BY SAGE Identification of differentially expressed

    genes in samples from different

    physiological or pathological conditions.Application of many statistical methods

    Poisson approximation

    Bayesian methodChi square test.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    36/64

    SAGE software searches GenBank for matchesto each tag

    This allows assignment to 3 categories of tags:

    mRNAs derived from known genes

    anonymous mRNAs, also known as expressed sequence

    tags (ESTs)

    mRNAs derived from currently unidentified genes

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    37/64

    SAGE VSMICROARRAY

    SAGEAn open system which detects both known and

    unknown transcripts and genes.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    38/64

    COMPARISON

    SAGE

    Detects 3 region of

    transcript. Restriction site

    is determining factor.

    Collects sequence

    information and copy no.

    Sequencing error and

    quantitation bias.

    MICROARRAY

    Targets various regions of

    the transcript.Base

    composition for specificity

    of hybridization.

    Fluorescent signals and

    signal intensity.

    Labeling bias and noise

    signals.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    39/64

    Contd

    Features SAGE M icroarray

    Detects unknown

    transcripts

    Yes No

    Quantification Absolute measure Relative measure

    Sensitivity High Moderate

    Specificity Moderate High

    Reproducibility Good for higher

    abundance transcripts

    Good for data from

    intra-platformcomparison

    Direct cost 5-10X higher than

    arrays.

    5-10 X lower than

    SAGE

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    40/64

    RECENT SAGE APPLICATIONS

    Analysis of yeast transcriptome

    Gene Expression Profiles in Normal and Cancer Cell

    Insights into p53-mediated apoptosis

    Identification and classification of p53-regulated genesAnalysis of human transcriptomes

    Serial microanalysis of renal transcriptomes

    Genes Expressed in Human Tumor Endothelium

    Analysis of colorectal metastases (PRL-3)Characterization of gene expression in colorectal adenomas

    and cancer

    Using the transcriptome to analyze the genome (Long SAGE)

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    41/64

    LIMITATIONS

    Does not measure the actual expression level of a gene.

    Average size of a tag produced during SAGE analysis is

    ten bases and this makes it difficult to assign a tag to a

    specific transcript with accuracy

    Two different genes could have the same tag and the same

    gene that is alternatively spliced could have different tags at

    the 3' ends

    Assigning each tag to an mRNA transcript could be madeeven more difficult and ambiguous if sequencing errors are

    also introduced in the process

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    42/64

    Quantitation bias: Contamination of of large quantities of linker-dimer molecules.

    low efficiency in blunt end ligation.

    Amplification bias.

    Depending upon anchoring enzyme and tagging enzyme

    used, some fraction of mRNA species would be lost.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    43/64

    Advances over SAGE

    Generation of longer 3` cDNA from SAGE tags

    for gene identification (GLGI)

    Long SAGE

    Cap Analysis of Gene Expression (CAGE)

    Gene Identification Signature (GIS)

    SuperSAGE

    Digital karyotyping

    Paired-end ditag

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    44/64

    Long SAGE

    Increased specificity of SAGE tags for

    transcript identification and SAGE tag

    mapping. Collects tags of 21bp

    Different TypeII restriction enzyme-Mmel

    Adapts SAGE principle to genomic DNA.

    Allows localisation of TIS and PAS.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    45/64

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    46/64

    CAGE (Capped Analysis of Gene Expression)

    Aims to identify TIS and promoters.

    Collects 21 bp from 5 ends of cap purified cDNA.

    Used in mouse and human transcriptome studies. The method essentially uses full-length

    cDNAs , to the 5ends of which linkers are

    attached.

    This is followed by the cleavage of the first 20

    base pairs by class II restriction enzymes,

    PCR, concatamerization, and cloning of the

    CAGE tags

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    47/64

    AAAAA

    AAAAABiotin

    Biotin +

    Mmel

    xBiotin

    +Xma JI

    Biotin

    Biotin Mmel-PCR

    BiotinUni-PCR

    XmaJI tag1 tag2 XmaJI

    Concatenation

    Cloning

    Sequencing

    PCR amplification

    Ligation to second linker

    MmeI digestion of dsDN

    ssDNA captureSecond strand synthesi

    Full strand DNA synthesis

    ssDNA release

    Reverse transcription

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    48/64

    Micro SAGE

    Requires 500-5000 fold less starting input RNA.

    Simplifies by the incorporation of a one tube procedure

    for all steps.

    Characterization of expression profiles in tissue biopsies,

    tumor metastases or in cases where tissue is scarce.

    Generation of region-specific expression profiles of

    complex heterogeneous tissues.

    Limited number of additional PCR cycles are performed to

    generate sufficient ditag.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    49/64

    An expression profile can be obtained from as

    little as 1-5 ng of mRNA.

    Comparison between the twoSAGE MicroSAGE

    Amount of input

    material

    2.5-5 ug RNA 1-5 ng of mRNA

    Capture of

    cDNA

    Streptavidin coated

    magnetic beads

    Streptavidin coated PCR

    tube

    Multiple tube vs.

    Single tube

    reaction

    Subsequent reactions in

    multiple tubes

    Multiple PCI extraction

    and ethanol precipitation

    steps

    Single tube reaction

    Easy change of buffers

    No PCI extraction or

    ethanol ppt step.

    Fewer manipulations

    PCR 25-28 cycles 28 cycles followed by re-PCR on excised ditag (8-

    15)

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    50/64

    SuperSAGE

    Increases the specificity of SAGE tags and

    use of tags as microarray probes.

    Type III REEcoP15Itag releasing

    Collects 26 bp tags

    Has been used in plant SAGE studies.

    Study of gene expression in which sequence

    information is not available.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    51/64

    Flowchart of superSAGE

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    52/64

    Gene Identification Signature

    (GIS) Identifies gene boundaries.

    Collects 20bp LongSAGE tags from 3 and

    5 end of the transcript.

    Applied to human and mouse transcription

    studies.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    53/64

    DIGITAL KARYOTYPING

    Analyses gene structure. Identification amplification and deletion in several

    cancers.

    PAIRED END DITAG

    Identifies protein binding sites in genome.

    Applied to identify p-53 binding sites in thehuman genome.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    54/64

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    55/64

    1. SAGE: A LOOKING GLASS

    FOR CANCER Deciphering pathways involved in tumor genesisand identifying novel

    diagnostic tools, prognostic markers,and potential therapeutic targets.

    SAGE is one of the techniques

    used in the National Cancer Institutefunded Cancer GenomeAnatomy Project (CGAP).

    A database with archived SAGE tag counts and on-line query toolswas

    created - the largest source of public SAGE data.

    More than 3 million tags from 88 different librarieshave been

    deposited on the National Center for BiotechnologyEducation/CGAP

    SAGEmap web site (http://www.ncbi.nlm.nih.gov/SAGE/).

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    56/64

    Several interesting patterns have emerged.

    cancerous and normal cells derived from the same tissue typeare verysimilar.

    tumors of the same tissue of origin but of different

    histological type orgrade have distinct gene expression patterns

    cancer cells usuallyincrease the expression of genes associated withproliferationand survival and decrease the expression of genes involved in

    differentiation.

    SAGE studies have been performed in patientswith colon, pancreatic,lung, bladder, ovarian, and breast cancers.

    SAGE experiments validated in multiple tumor and normaltissue pairsusing a variety of approaches, including Northernblot analysis, real-

    time PCR, mRNA in situ hybridization, and

    immunohistochemistry.

    Identification of an ideal tumor marker. E.g. Matrix metalloprotease1in ovarian cancer is overexpressed.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    57/64

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    58/64

    p53- TUMOR SUPRESSOR GENE

    p53is thought to play a rolein the regulation of cell cycle checkpoints,apoptosis, genomicstability, and angiogenesis.

    Sequence-specific transactivationis essential forp53-mediated tumorsuppression.

    The analysis of transcriptomes afterp53expressionhas determinedthatp53exerts its diverse cellular functionsby influencing theexpression of a large group of genes.

    Identification of Previously Unidentified p53-Regulated Genes bySAGE analysis.

    Variability exists with regardto the extent, timing, andp53dependenceof the expressionof these genes.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    59/64

    2. IMMUNOLOGICAL STUDIES

    Only a few SAGE analysis has been applied for the study ofimmunological phenomena.

    SAGE analyses were conducted for human monocytes and their

    differentiated descendants, macrophages and dendritic cells.

    DC cDNA library represented more than 17,000 different genes. Genesdifferentially expressed were those encoding proteins related to cellmotility and structure.

    SAGE has been applied to B cell lymphomas to analyze genesinvolved in BCRmediated apoptosis.- polyamine regulation isinvolved in apoptosis during B cell clonal deletion.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    60/64

    Contd

    LongSAGE has been used to identify genes of T cells with SLE that

    determine commitment to the disease.

    Findings indicate that the immatureCD4+ T lymphocytes may be

    responsible for the pathogenesis of SLE.

    SAGE has been used to analyze the expression profiles of Th-1 and Th-

    2 cells, and newly identified numerous genes for which expression is

    selective in either population.

    Contributes to understanding of the molecular basis of Th1/Th2

    dominated diseases and diagnosis of these diseases.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    61/64

    3. YEAST TRANSCRIPTOME

    Yeast is widely used to clarify the biochemical physiologicparameters underlying eukaryotic cellular functions.

    Yeast chosen as a model organism to evaluate the powerof SAGE technology.

    Most extensive SAGE profile was made for yeast.

    Analysis of yeast transcriptome affords a unique view ofthe RNA components defining cellular life.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    62/64

    4.ANALYSIS OF TISSUE

    TRANSCRIPTOMES

    Used to analyze the transcriptomes of renal, cervical

    tissues etc.

    Establishing a baseline of gene expression in normal tissue

    is key for identifying changes in cancer.

    Specific gene expression profiles were obtained, and

    known markers (e.g., uromodulinin the thick ascending

    limb of Henle's loop and aquaporin-2 inthe collecting duct)

    were found.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    63/64

    REFERENCES

    Maillard, Jean-Charles, et al., Efficiency and limits of the Serial Analysis ofGene Expression., Veterinary Immunol. and Immunopathol. 2005., 108:59-69.

    Man, M.Z. et al., POWER-SAGE: comparing statistical tests for SAGEexperiments., Bioinformatics 2000., 16: 953-959.

    Polyak, K. and Riggins, G.J., Gene discovery using the serial analysis of geneexpression technique: Implications for cancer research., J. of Clin. Oncol.2001., 19(11):2948-2958.

    Tuteja and Tuteja., Serial Analysis of Gene Expression: Applications inHuman Studies., J. of Biomed. And Biotechnol. 2004., 2: 113-120.

    Tuteja and Tuteja., Serial analysis of gene expression: application in cancerresearch., Med. Sci. Monit. 2004., 10(6): 132-140.

    Velculescu, V.E. et al. Serial analysis of gene expression., Science 1995.,

    270:484-487. Wing, San Ming., Understanding SAGE data., Trends in Genetics 2006., 23:

    1-12.

    Yamamoto, M., et al., Use of serial analysis of gene expression (SAGE)technology., J. of Immunol. meth.2001., 250:45-66.

  • 8/12/2019 ``SAGE 10 - Includes Advances of SAGE

    64/64