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Roche/454 Genome Sequencing
The 454 Genome Sequencer FLX Instrument, powered by GS FLX Titanium chemistry,features a groundbreaking combination of long reads (300-500bp), exceptional accuracy, and high throughput. The instrument uses a system based on 454's sequencingby-synthesis technology.
The high throughput 454/GS FLX Titanium sequencing technology has many applications.Some of the most common are: • Whole genome sequencing (de novo, resequencing) via: o Shotgun sequencing (bacteria, viruses) and o BAC-based shotgun sequencing (animals, plants). Amplicon sequencing - newly released with Titanium chemistry! Small RNA sequencing, including expressed sequence tags (ESTs) o Assembled de novo for novel organisms or o Mapped to genome or transcriptome references for characterized organisms Transcriptome analysis
**Note: We are a facility dedicated to helping researchers achieve their goals. If there is a specific application you are interested in but do not see listed here, please do not hesitate to contact us to see if we can accomodate your needs!
Accepted Sample Types
• • • • Genomic DNA PCR products BACs cDNAs of all sizes
• • • • • • DNA must be double stranded. DNA should not be the result of whole genome amplification (or other similar process which may compromise representativity) DNA should not be degraded, i.e. starting DNA material should be in pieces >1.5kb (70-800bp for LMW DNA) DNA should contain no particulate matter DNA should have an OD 260/280 ratio ≥ 1.8 Total DNA required:
5kb) are nebulized into fragments 300 to 800 . How Pyrosequencing Works: Overview The complete sequencing workflow of the Genome Sequencer FLX System is comprised of four main steps: 1. but is not recommended. Samples may be left in the freezer at the north entrance to our lab. BACs and cDNA libraries (larger than ~1. room 124. Shipping address: University of Arizona Genetics Core Attn: 454 Sequencing 1657 E. so please do not ship packages for delivery those days unless you would like it to sit on the loading dock until Monday morning (not the best idea in the Arizona heat!). University customers: we are located in the Keating Building. we will have to dry the sample down and re-suspend using ultra-pure Milli-Q water.• o 700ng-1µg total per sample DNA sample should be suspended in ≤ 100µL TE o If the sample is in a volume >100µL. clearly labeled "454 Sequencing" along with your contact information and billing account. 2. 4. 3. Large samples such as genomic DNA. ensuring that there is minimal degradation.Customers will be asked for more DNA if their sample fails either of the following check points: • • A PicoGreen Assay to verify the amount of starting DNA A Bioanalyzer DNA 7500 chip to check the quality of DNA. AZ 85721 Quality Control of Samples Every sample received for sequencing will go through a set of quality control checks before it can be processed. This does usually not result in significant degradation. External customers: samples should be shipped pre-frozen and prefferably on dry ice (although regular ice packs work well enough for overnight shipping). Helen Street Keating Building. Rm 124 Tucson. We do not have the availability to receive packages on Saturdays or Sundays. Generation of a template DNA library Emulsion-based clonal amplification of the library Data generation via sequencing-by-synthesis Data analysis using different bioinformatics tools Library Preparation Short PCR products amplified using Roche/454 fusion primers do not need to undergo the library preparation process and can be used directly for immobilization onto DNA capture beads.
The adaptors enable subsequent amplification. This release of light is recorded by an extremely sensitive CCD camera. the sample library is double-stranded. The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads. fragmentation is not required.$5.360 flat rate per whole plate Sequencing .specific for both the 3' and 5' ends .$530 per sample Titrations . Using a series of standard molecular biology techniques. short adaptors (A and B) . Library Preparation .Cost will vary depending on project requirements.0% surcharge • • • • • Expected Results Titanium GS FLX Chemistry: . The diameter of the wells on a PicoTiterPlate allow only one DNAcontaining bead to be deposited per well. this results in a copy number of several million per bead. Following amplification. The fluidics subsystem of the Genome Sequencer FLX Instrument flows individual nucleotides in a fixed order across the entire plate.820 flat rate per whole plate External users will incur a 11. The bead-bound library is emulsified in a water-in-oil mixturewith amplification enzymes and primers.$5.basepairs in length. surrounded by much smaller beads with attached sulphurylase and luciferase. such as small non-coding RNA or PCR amplicons. ideally so that each bead carries a unique singlestranded DNA library fragment. Emulsion PCR (emPCR) Amplification Each unique sample library fragment is amplified within its own microreactor. and for homopolymer repeats (multiple incorporations of the same nucleotide) up to six nucleotides. single-stranded fragments with A and B adaptors comprise the sample library. Pricing for Titanium GS FLX Chemistry: • Sample Preparation . and sequencing steps. excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel. An experimentallydetermined volume of library (see "Titration") is added to the capture beads. the emulsion is broken and excess enzymes/oil/primer are washed away while the amplified fragments remain bound to their specific beads. o Note that this charge does not apply for most samples. converting to ATP and producing light from the oxidation of luciferin to oxyluciferin. For smaller samples.$990 per sample Bulk emPCR . Sequencing The clonally amplified fragments are enriched and loaded onto a PicoTiterPlate device for sequencing (approximately 1 million beads are deposited onto one region of a 2-region plate). the number of bases added is directly proportional to the light signal detected. Double-stranded libraries prepared with the rapid library technique undergo a denaturing step before the fragments can be captured. for each fragment. purification. Please contact us for more information regarding our sample preparation services. resulting in microreactors surrounding only one bead with one unique sample-library fragment.are ligated to the fragmented DNA. After general library preparation. Following a rapid library preparation. You will only pay a sample preparation charge if we are synthesizing cDNA from RNA or extracting the genetic material of interest for you. Addition of a nucleotide complementary to the template strand results in a release of one pyrophosphate unit.
php/next-gen-sequencing-services/roche454.arizona. Average length = 400 bases Accuracy Q20 read length of 400 bases (99% at 400 bases and higher for prior bases) Reads per run: >1 million high-quality reads Forms: • • 454 De Novo Contig Assembly form 454 Data Processing for Mapping to Reference Sequences http://uagc.edu/index.html The development and impact of 454 sequencing .arl. filter-passed bases per run Read Length: Modal length = 500 bases. filter-passed bases per run Throughput for 4-Region Plate: 240-440 million high-quality.Throughput for 2-Region Plate: 360-560 million high-quality.
and more.(a) Genomic DNA is isolated. a flow cell that includes the well-containing fiber-optic slide (object ii). a CCD camera-based imaging assembly with its own fiber-optic bundle used to image the fiber-optic slide (part of object iii). ligated to adapters and separated into single strands.gif ILLUMINA SEQUENCING: Chemistry for Sequencing Illumina’s sequencing by synthesis (SBS) technology is the most successful and widelyadopted next-generation sequencing platform worldwide.ilmn . showing fiberoptic cladding and wells before bead deposition. the DNA strands are denatured. including: whole-genome and candidate region resequencing. fragmented. natural competition minimizes incorporation bias. http://www. http://www. transcriptome analysis. resulting in beads each carrying ten million copies of a unique DNA template. See a video of Illumina sequencing technology in action. large-scale structural variation detection. and a computer that provides the necessary user interface and instrument control (part of object iii). small RNA discovery. the beads are isolated and compartmentalized in the droplets of a PCR-reaction-mixture-in-oil emulsion and PCR amplification occurs within each droplet. (e) Scanning electron micrograph of a portion of a fiber-optic slide.com/nbt/journal/v26/n10/images/nbt1485-F2. (d) Smaller beads carrying immobilized enzymes required for a solid phase pyrophosphate sequencing reaction are deposited into each well. and genome-wide protein-nucleic acid interaction analysis. Applications for Sequencing SBS technology supports both single read and paired-end libraries. de novo sequencing.illumina. A fluorescently-labeled terminator is imaged as each dNTP is added and then cleaved to allow incorporation of the next base. Since all four reversible terminator-bound dNTPs are present during each sequencing cycle.nature. The combination of short inserts and longer reads increase the ability to fully characterize any genome. A wide array of available sample preparation methods serve to enable diverse applications. The end result is true base-by-base sequencing that enables the industry’s most accurate data for a broad range of applications. and beads carrying single-stranded DNA templates are enriched (not shown) and deposited into wells of a fiber-optic slide. It is the only platform that offers a short-insert paired-end capability for high-resolution genome sequencing as well as long-insert paired-end reads using the same robust chemistry for efficient sequence assembly. (c) The emulsion is broken. (b) Fragments are bound to beads under conditions that favor one fragment per bead. (f) The 454 sequencing instrument consists of the following major subsystems: a fluidic assembly (object i). TruSeq technology supports massively parallel sequencing using a proprietary reversible terminator-based method that enables detection of single bases as they are incorporated into growing DNA strands.com/technology/sequencing_technology. methylation profiling.
http://seqanswers. . 3. After PCR.jpg SOLID SEQUENCING : Library Preparation 1. beads. Emulsion PCR/Bead Enrichment 2. denature the templates and perform bead enrichment to separate beads with extended templates from undesired beads. Your choice of library depends on the application you're performing and the information you desire from your experiments. PCR reaction components. Prepare clonal bead populations (Figure 2) in microreactors containing template. The template on the selected beads undergoes a 3’ modification to allow covalent attachment to the slide.com/forums/images/content/ilmn-step1-6. and primers. Prepare one of the two types of libraries (Figure 1) for SOLiD™ System sequencing-fragment or mate-paired.
or eight sections. A set of four fluorescently labeled di-base probes compete for ligation to the sequencing primer.Bead Deposition 4. four. deposition chambers enable you to segment a slide into one. A key advantage of the system is the ability to accommodate increasing densities of beads per slide. 6. During bead loading. resulting in a higher level of throughput from the same system. Deposit 3’ modified beads onto a glass slide (Figure 3). Primers hybridize to the P1 adapter sequence on the templated beads (Figure 4). Sequencing by Ligation 5. Specificity of the di-base probe .
7. detection and cleavage are performed with the number of cycles determining the eventual read length. Primer Reset . the extension product is removed and the template is reset with a primer complementary to the n-1 position for a second round of ligation cycles.is achieved by interrogating every 1st and 2nd base in each ligation reaction. Multiple cycles of ligation. Following a series of ligation cycles. 8.
This dual interrogation is fundamental to the unmatched accuracy characterized by the SOLiD™ System. virtually every base is interrogated in two independent ligation reactions by two different primers.99% accuracy is achieved with the Exact Call Chemistry Module by sequencing with an additional primer using a multi-base encoding scheme. Through the primer reset process.9. the base at read position 5 is assayed by primer number 2 in ligation cycle 2 and by primer number 3 in ligation cycle 1 (see figure at right).Up to 99. Five rounds of primer reset are completed for each sequence tag (Figure 5). For example. Exact Call Chemistry 10. THIRD GENERATION: Ion Torrent .
When a polymerase incorporates a nucleotide into a strand of DNA. Whole transcriptome: o 125-625ng of poly(A) RNA or 250-625ng of rRNA-depleted total RNA o Suspended in 10uL Nuclease-free water o Absent of contaminating rRNA Quality Control of Samples: Every sample received for sequencing will go through a set of quality control checks before it can be processed. Applications: • • • • Small Genome Sequencingo Microbial and viral de novo & resequencing Targeted Resequencing (Amplicon) Whole Genome/Exome Validation o validate other platform results RNA Sequencing o Whole Transcriptome Accepted Sample Types: • • • • • Genomic DNA PCR product BACs cDNA RNA Sample Requirements: DNA Sample requirements- • • • • • • • DNA must be double stranded DNA should not be the result of whole genome amplification (or other similar process which may compromise representativity) DNA should not be degraded DNA should contain no particulate matter DNA should have an OD 260/280 ratio ≥ 1. Customers will be asked for more DNA/RNA if their sample fails either of the following check points: . or optics. Ion Torrent captures this process in a massively parallel way using DNA loaded Ion SpheresTM on a high density array of wells atop a proprietary Ion sensor. a hydrogen ion is released resulting in a pH change.8 5µg total DNA required DNA sample should be suspended in ≤ 130µL TE RNA Sample Requirements- • • • RNA should not be degraded RNA should have an RNA integrity number (RIN) greater than 7.The Ion Torrent Personal Genome Machine (PGM) pairs natural sequencing chemistry with semiconductor technology resulting in a revolutionary sequencing process without fluorescence. enzymatic cascades.
edu prior to sample submission. External customers: Shipping Guidelines- • • • • • Samples should be shipped in screw-cap or snap-cap centrifuge tubes secured with Parafilm. 100bp average length Accuracy: >99.arl. We do not have the availability to receive packages on Saturdays or Sundays.arizona. 100bp average length 316 Chip: 100Mbps output. and basic first pass assembly or mapping using Roche 454 software. http://uagc.arizona. ensuring that there is minimal degradation. AZ 85721 Expected Results: • • • 314 Chip: 10Mb output.99% consensus and >99.edu/index. Shipping address: University of Arizona Genetics Core Attn: Ion Torrent Sequencing 1657 E. Sample Submission: Please contact Christine Schirmer email@example.com/next-gen-sequencing-services/ion-torrent.html . Samples should be placed in a box or a larger tube padded with Kimwipes.6% raw. Helen Street Keating Building. Samples should be shipped pre-frozen and on dry ice (regular ice packs are okay for overnight shipping of DNA ONLY). University customers: We are located in the Keating Building. Please email tracking number. 124 Tucson. Please contact Christine Schirmer to arrange for sample drop-off. Rm.• • Pico/RiboGreen Assay to verify the amount of starting material Bioanalyzer DNA/RNAchip to check the quality of DNA/RNA. room 124. Data Analysis: Sequencing service includes raw sequence files.
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