nrg2612-s1 by dandanhuanghuang


									                                        Techonology                              Compagnies                                 Specifications                                       pros                                             cons                            REF        Cost
                                                                                                                 read length up to 500 bp
                                                                                                                                                                                                               difficult to control variance in coverage, SNP
                                          Pyrosequencing                                 454                   Total bp yield per run: 500 Mb                               long read length                                                                     [XX],[XX]    $$
                                                                                                                                                                                                                vs calling error, difficulties with homo runs
                                                                                                                               Run time : 10 hours
                                                                                                                  read length up to 65 bp
                                      Sequence by synthesis                            Illunima                  Total bp yield per run: 3Gb                       Cost effective/Mp, short runtime                         Short read length,                   [XX],[XX]    $$
                                                                                                                               Run time : 3 days
 High throughput sequencing                                                                                    read length up to 40 bp
                                          Ligation based                             ABI solidTM               Total bp yield per run: 20Gb                      Most acquirate, proof reading system                          Long run time                     [XX],[XX]    $$
                                                                                                                                 Run time : 5 days
                                                                                                                read length up to 30-50 bp
                                    Single molecule sequencing                Pacific bioscience, Helicos        Total bp yield per run:                                     no pcr biased                               Technology at its infancy               [XX],[XX]    $$
                                                                                                                                   Run time:

                                                                                                                   Cost effecitive range:
                                        Hybridization based                    Affymetrix, Nimblegen                                                                 Very cost effective for GWAS                         Not easily costumized                  [XX],[XX]    $$
                                                                                                                          Available for GWAS: Yes

                                                                                                                   Cost effecitive range:
        Genotyping                   Single bp extension assays                        Illunima
                                                                                                                          Available for GWAS: Yes
                                                                                                                                                                     Very cost effective for GWAS                         Not easily costumized                  [XX],[XX]    $$

                                                                                                                   Cost effecitive range: 100 - 10,000 SNP
                                                                                     Sequenom                                                                   Very cost effective for target SNP arrays                     SNP resolution                     [XX],[XX]    $$
                                                                                                                           Available for GWAS: Yes

                                                                                                                                                                                                                  Rely on existing genomic sequences,
                                                                                                                    hybridization based
                                         cDNA microarrays                              Agilent                                                                          low cost, high througput               hybridization background effect, biased by        [XX],[XX]    $$
                                                                                                                        resolution: limited to exon
                                                                                                                                                                                                                 gene model, often low dynamic range,
                                                                                                                                                                                                                  Rely on existing genomic sequences,
                                                                                                                   hybridization based
                                           tilling arrays                            Affymetrix                                                              Not biased by a gene model, high throughput        hybridization background effect, limited         [XX],[XX]    $$
                                                                                                                            reslution: custumized
                                                                                                                                                                                                                     ability to call allelic expression,
Whole genome transcriptional
         profiling                                                                                                                                               For some application - Does not rely on
                                                                                                                                                             existing genomic sequences, no normalization
                                                                                                                   hybridization based                        problems or background effects, not biased        Difficulies is mapping sequence tag to the
                                             RNA seq                           454, ABI solid, Illumina                                                                                                                                                          [XX],[XX]    $$
                                                                                                                            reslution: unlimited              by gene models, relatively low cost, requires                    genomic region.
                                                                                                                                                               low amount of RNA, can distinguish allelic
                                                                                                                                                                  expression, very large dynamic range

                                                                                                                                                              High resolution, unbiased, provide peptide       Expensive, profile can be difficult to match
                                LC-ESI-MS/MS and Tandem MS/MS          MALDI-ToF Pro by Amersham Biosciences                                                                                                                                                     [XX],[XX]    $$
                                                                                                                                                                               sequence                                   with know molecules

                                                                                                                                                                                                               Limited number of candidate protein can be
         Proteomics                   Protein bio-chip arrays                          Randox                                                                       High throughput, very sensitive               assayed one. Rely on protein specific          [XX],[XX]    $$

                                                                                                                                                                                                              Difficulties with the analysis (matching protein
                                  Two dimensional gel separation                      Invitrogen                                                                     Inexpensive and quantitative                                                                [XX],[XX]    $$
                                                                                                                                                                                                                          fragments across samples)

                                   mass spectrometry FT-ICR-MS                                                                                                                                                                                                   [XX],[XX]    $$

       Metabolomics                 nuclear magnetic resonance                                                                                                                                                                                                   [XX],[XX]    $$

                               Capillary gas chromatography–time-of-
                                                                                                                                                                                                                                                                 [XX],[XX]    $$
                                flight–mass spectrometry GC-TOF-MS

                                      Raman spectroscopies                                                                                                                                                                                                       [XX],[XX]    $$

                                       DIESI direct-infusion                                                                                                                                                                                                     [XX],[XX]    $$

                                                                                     Ethovision                                                                                                                                                                  [XX],[XX]    $$

High throughput phenotyping
                                                                                                                                                                                                                                                                 [XX],[XX]    $$
Supplementary Table 1 | Technologies enabling systems genetics
Read length is long
                                                  Technology                                                       Description                                                     Companies                         Specifications

                                                                         Fragmented DNA is ligated to short oligo adaptors and fixed onto beads where a PCR reaction
                                                                         amplifies the fragments. As in pyrosequencing, the release of a pyrophosphate molecule during
                                                                                                                                                                                                         Read length up to 500 bp
                                                                         the incorporation of nucleotides by DNA polemerase is used to produce a nucleotide specific
                                                  Pyrosequencing                                                                                                                          454            Total bp yield per run: 500 Mb
                                                                         fluorescence which is sequentially recorded by a camera, allowing the sequencing of the fragment.
                                                                                                                                                                                                                         Run time : 10 hours
                                                                         The long reads produced by this technology make it particularly appropriate for de novo
                                                                         assembly but the low coverage makes it one of the most expensive methods.

                                                                         The DNA template is initially immobilized by ligation to adaptors which are bound to a slide. This
                                                                         technology relies on the modification of the dideoxynucleotide terminator used in Sanger
                                                                         sequencing. While normally the incorporation of a ddNTP permanently prevents DNA polymerase                                     Read length up to 75 bp
                                                                                                                                                                                   Illumina Genome
                                               Sequence by synthesis     from adding additional nucleotides, Illumina engineered a reversible version of the terminator.                                 Total bp yield per run: 3Gb
                                                                         Each modified dNTP is bound to a base-specific fluorophore which flouresces when incorporated                                                 Run time : 3 days
                                                                         into the DNA fragment. The emission is recorded by a high-resolution camera. In each cycle, a
                                                                         labeled dNTP is incorporated, a picture is taken, and the terminator is removed.

                      High-throughput                                    This technology does not rely on polymerase-driven incorporation of labeled ddNTP terminators.
                                                                         Instead, after the DNA template has been fragmented, amplified, ligated to adaptors and
                         sequencing                                      immobilized on a glass slide. Primers are hybridized to an adapter sequence and four
                                                                                                                                                                                                         Read length up to 40 bp
                                                   Ligation based                                                                                                                     ABI SOLiDTM         Total bp yield per run: 5Gb
                                                                         fluorescently labeled dinucleotide probes compete for ligation. Following detection, cleavage
                                                                                                                                                                                                                          Run time : 5 days
                                                                         occurs, and the cycle repeats as the read is extended. The extension product is removed and the
                                                                         template is reset with a new primer in each of five rounds to ensure accuracy.

                                                                         This approach does not require any amplification of the DNA template. In the case of Pacific
                                                                                                                                                                                                            Read length is long
                                                                         Biosciences, DNA polymerase is fixed in microscopic wells. The dNTPs used in this reaction are
                                                  Single molecule                                                                                                                 Pacific Biosciences,     Total bp yield per run is unrestricted but
                                                                         bound to base-specific fluorophores. As the polymerase adds dNTPs to the template, the
                                                     sequencing                                                                                                                          Helicos         highly variable
                                                                         fluorophores are cleaved. Cleavage at each cycle produces a nucleotide-specific fluorescence
                                                                                                                                                                                                                      Run time is a few hours
                                                                         which is recorded to form the DNA sequence.

                                                                          A genomic region of interest (e.q. a large QTL region ) is isolated either by hybridation to a tiling
                                                                         array or via a series of long-range PCRs. The resulting fragments are then analyzed using one of
                                                                                                                                                                                                              Relies on long range PCR or array
                                                                         the deep sequencing technologies described aboved. The approach can be cost effective because
                                                 Sequence capture                                                                                                                 Agilent, Nimblegen         hybridization followed by any of the
                                                                         indvidual-specific sequence tags can be ligated to the DNA fragments of each individual, allowing
                                                                                                                                                                                                                      technologies above
                                                                         DNA fragments from different individuals to be pooled during the sequencing reaction. The
                                                                         ligated tags can then be used to identify the origin of the sequences.

                                                                         Genotypes are distingusihed on the basis of differential hybridization to a given probe sequence                                   Typical use is from a few hundred to a
                                                Hybridization based      (also called a feature). Probes can be allele-specific, facilitating the identification SNP-specific                            thousand markers                    Whole-
                                                                         differences.                                                                                                                           genome genotyping available

                        Genotyping                                       For each polymorphism to be genotyped, allele-specific oligos (ASO) are synthesized and paired
                                                                         with either Cy3 or Cy5 dye. After the DNA template is immobilized on beads, these oligos are
                                                Single bp extension                                                                                                                Illunima Golden       Typical use varies by platform
                                                                         hybridized. When the ASO and DNA sequences are complementary, DNA polymerase catalyzes an
                                                       assays                                                                                                                          Gate assay            Whole-genome genotyping available
                                                                         allele-specific extension. Upon completion of this process a fluorescent signal is produced for
                                                                         each allele, and the relative intensity of the dyes is translated into a genotype call.

                                                                         DNA oligonucleotides complementary to specific gene sequences are synthesized and printed
                                                                                                                                                                                                          Hybridization based
                                                                         onto a glass surface. These oligos are then used as probes to hybridize fluorescently labeled cDNA
                                                                                                                                                                                  Agilent, Nimblegen, Resolution ususally limited to exons
                                                 cDNA microarrays        or cRNA. Fluorescence levels of each probe after hybridization are used to measure the expression
                                                                                                                                                                                  Ilumina, Affymetrix            Probe length varies from 25bp to
                                                                         level of each gene. Allows researchers to monitor the expression level of thousands of gene
                                                                                                                                                                                                                    several hundred
                                                                         simultaneously at a low cost.

                      Whole-genome                                       Similar to cDNA microarrays except that the short oligonucleotide probes are designed to densely
                                                                                                                                                                                                           Hybridization based
                      transcriptional                                    span a genomic region (possibly the entire genome). Probes can be overlapping or spaced                  Affymetrix, Agilent,
                                                    Tiling arrays                                                                                                                                        Resolution varies                    Probe
                                                                         regularly. Tiling arrays can be used to identify novel non-exonic transcripts or to study alternative        Nimblegen
                         profiling                                                                                                                                                                             length usually from 25 to 70 bp

                                                                         Leverages any of the 'deep sequencing' technologies described above to perform an unbiased                                          Specifications depend on sequencing
                                                                         screen of the transcriptome. Total or mRNA is extracted and sequenced as DNA would be. The                 454, ABI solid,      technology used. Aligning RNA sequence to
                                                      RNA seq
                                                                         resulting sequences are aligned to the genome and coverage is used a measure of the expression                Illumina          a genome is challenging when fragments are
                                                                         level for that region.                                                                                                                            very short.

                                                                         A mixture of proteins are digested into short peptides to be separated using liquid                      MALDI-ToF Pro by        Several hundred proteins can be identified
                                               Tandem MS/MS and
                                                                         chromatography (see below). The fragments are analyzed using tandem mass spectrometry (also                Amersham               though this method               Largely
                                               Shot gun proteomics
                                                                         called MS/MS) which allows the sequencing of these peptides. Th                                            Biosciences                          qualitative

                                                                         Analogous to microarrays, protein-specific antibodies are generated, printed on a glass side, and
                                               Protein bio-chip arrays                                                                                                                  Randox              Limited throughput, data can be noisy
                                                                         used as probes to detect and quantify the amount of fluorescently labeled proteins in the sample.

                                                                         Straightforward approach to identify post-transcriptional differences between samples. Proteins
                                                                         are initially separated based on their mass on a gel; subsequently, the gel is rotated and proteins
                                               Two dimensional gel
                                                                         are separated based on their isoelectric point. Mixtures of proteins from two samples can be run             Invitrogen            Limited throughput, data can be noisy
                                                                         simultaneously on the same gel and labeled with different dyes to allow the identification of
                                                                         differences between the samples. Proteins can then be extracted from the gel for further analysis.

                                                                     Metabolites are separated by weight and change using either gas chromatography (GC) or high-
                                                                     performance liquid chromotography (HPLC). Subsequently, MS ionizes these compunds to
                                                                     produce charged molecules whose mass-to-charge ratio can be measured as a molecule-specific                     PerkinElmer,        Most commonly used methods can identify
                                              Mass Spectrometry (MS)
                                                                     signature. Mass-to-charge ratio profiles are compared to a library of molecule-specific profiles,                 Thermos                  several hundred metabolites
                                                                     allowing the identification of various metabolites. MS is most commonly used in combination with
                      Metabolomics                                   GC.

                                                                                                                                                                                                         Not as prevalent as MS despite the fact that
                                                                         This approach relies on the fact that different molecules have a differrent reasonance frequency
                                                 Nuclear magnetic                                                                                                                                           separation is not required
                                                                         when a magnetic field is applied. This frequency can be recorded and, as in MS, the resulting                  Varian
                                                 resonance (NMR)                                                                                                                                          Sensitivity is lower and may be difficult to
                                                                         profiles are compared to a database of molecule-specific profiles for identification.
                                                                                                                                                                                                              match output with known profiles

                                               Image/video analysis
                                                                         Parallels the importance of high-throughput genotyping. High-throughput phenotypic allows for
                      High-throughput          based approaches, in
                                                                         faster, and sometimes more accurate data collection, thus accomodating the increased sample              Ethovision, Cadabra                   System specific
                        phenotyping              vivo physiological
                                                                         sizes that will be facilitated by high-throughput genotyping.
                                                    sensor, etc.

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