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] $$ sequence with know molecules Limited number of candidate protein can be Proteomics Protein bio-chip arrays Randox High throughput, very sensitive assayed one. Rely on protein specific [XX],[XX] $$ antibodies 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 Analyzer 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 Affymetrix, Hybridization based (also called a feature). Probes can be allele-specific, facilitating the identification SNP-specific thousand markers Whole- Nimblegen 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 splicing. 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. Proteomics 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 separation 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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