SECOND GENERATION:

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.

Applications
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

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, 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:

o 700ng-1g total per sample DNA sample should be suspended in 100L TE o If the sample is in a volume >100L, we will have to dry the sample down and re-suspend using ultra-pure Milli-Q water. This does usually not result in significant degradation, but is not recommended.

University customers: we are located in the Keating Building, room 124. Samples may be left in the freezer at the north entrance to our lab, clearly labeled "454 Sequencing" along with your contact information and billing account. External customers: samples should be shipped pre-frozen and prefferably on dry ice (although regular ice packs work well enough for overnight shipping). We do not have the availability to receive packages on Saturdays or Sundays, 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!).

Shipping address:
University of Arizona Genetics Core Attn: 454 Sequencing 1657 E. Helen Street Keating Building, Rm 124 Tucson, 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.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, ensuring that there is minimal degradation.

How Pyrosequencing Works:

Overview
The complete sequencing workflow of the Genome Sequencer FLX System is comprised of four main steps: 1. 2. 3. 4. 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. Large samples such as genomic DNA, BACs and cDNA libraries (larger than ~1.5kb) are nebulized into fragments 300 to 800

basepairs in length. For smaller samples, such as small non-coding RNA or PCR amplicons, fragmentation is not required. Using a series of standard molecular biology techniques, short adaptors (A and B) - specific for both the 3' and 5' ends - are ligated to the fragmented DNA. The adaptors enable subsequent amplification, purification, and sequencing steps. After general library preparation, single-stranded fragments with A and B adaptors comprise the sample library. Following a rapid library preparation, the sample library is double-stranded. The single-stranded DNA library is immobilized onto specifically designed DNA Capture Beads. Double-stranded libraries prepared with the rapid library technique undergo a denaturing step before the fragments can be captured. An experimentallydetermined volume of library (see "Titration") is added to the capture beads, ideally so that each bead carries a unique singlestranded DNA library fragment. The bead-bound library is emulsified in a water-in-oil mixturewith amplification enzymes and primers, resulting in microreactors surrounding only one bead with one unique sample-library fragment.

Emulsion PCR (emPCR) Amplification

Each unique sample library fragment is amplified within its own microreactor, excluding competing or contaminating sequences. Amplification of the entire fragment collection is done in parallel; for each fragment, this results in a copy number of several million per bead. Following amplification, the emulsion is broken and excess enzymes/oil/primer are washed away while the amplified fragments remain bound to their specific beads.

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 diameter of the wells on a PicoTiterPlate allow only one DNAcontaining bead to be deposited per well, surrounded by much smaller beads with attached sulphurylase and luciferase. The fluidics subsystem of the Genome Sequencer FLX Instrument flows individual nucleotides in a fixed order across the entire plate. Addition of a nucleotide complementary to the template strand results in a release of one pyrophosphate unit, converting to ATP and producing light from the oxidation of luciferin to oxyluciferin. This release of light is recorded by an extremely sensitive CCD camera, and for homopolymer repeats (multiple incorporations of the same nucleotide) up to six nucleotides, the number of bases added is directly proportional to the light signal detected.

Pricing for Titanium GS FLX Chemistry:

Sample Preparation - Cost will vary depending on project requirements. o Note that this charge does not apply for most samples. You will only pay a sample preparation charge if we are synthesizing cDNA from RNA or extracting the genetic material of interest for you. Please contact us for more information regarding our sample preparation services. Library Preparation - $530 per sample Titrations - $990 per sample Bulk emPCR - $5,360 flat rate per whole plate Sequencing - $5,820 flat rate per whole plate External users will incur a 11.0% surcharge

Expected Results
Titanium GS FLX Chemistry:

Throughput for 2-Region Plate: 360-560 million high-quality, filter-passed bases per run Throughput for 4-Region Plate: 240-440 million high-quality, filter-passed bases per run Read Length: Modal length = 500 bases, 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.arl.arizona.edu/index.php/next-gen-sequencing-services/roche454.html

The development and impact of 454 sequencing

(a) Genomic DNA is isolated, fragmented, ligated to adapters and separated into single strands. (b) Fragments are bound to beads under conditions that favor one fragment per bead, the beads are isolated and compartmentalized in the droplets of a PCR-reaction-mixture-in-oil emulsion and PCR amplification occurs within each droplet, resulting in beads each carrying ten million copies of a unique DNA template. (c) The emulsion is broken, the DNA strands are denatured, and beads carrying single-stranded DNA templates are enriched (not shown) and deposited into wells of a fiber-optic slide. (d) Smaller beads carrying immobilized enzymes required for a solid phase pyrophosphate sequencing reaction are deposited into each well. (e) Scanning electron micrograph of a portion of a fiber-optic slide, showing fiberoptic cladding and wells before bead deposition. (f) The 454 sequencing instrument consists of the following major subsystems: a fluidic assembly (object i), 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), and a computer that provides the necessary user interface and instrument control (part of object iii).

http://www.nature.com/nbt/journal/v26/n10/images/nbt1485-F2.gif
ILLUMINA SEQUENCING: Chemistry for Sequencing Illuminas sequencing by synthesis (SBS) technology is the most successful and widelyadopted next-generation sequencing platform worldwide. 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. 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, natural competition minimizes incorporation bias. The end result is true base-by-base sequencing that enables the industrys most accurate data for a broad range of applications. See a video of Illumina sequencing technology in action. Applications for Sequencing SBS technology supports both single read and paired-end libraries. 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, de novo sequencing, large-scale structural variation detection, and more. 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, including: whole-genome and candidate region resequencing, transcriptome analysis, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis.

http://www.illumina.com/technology/sequencing_technology.ilmn

http://seqanswers.com/forums/images/content/ilmn-step1-6.jpg SOLID SEQUENCING : Library Preparation 1. Prepare one of the two types of libraries (Figure 1) for SOLiD System sequencing-fragment or mate-paired. Your choice of library depends on the application you're performing and the information you desire from your experiments.

Emulsion PCR/Bead Enrichment 2. Prepare clonal bead populations (Figure 2) in microreactors containing template, PCR reaction components, beads, and primers. 3. After PCR, denature the templates and perform bead enrichment to separate beads with extended templates from undesired beads. The template on the selected beads undergoes a 3 modification to allow covalent attachment to the slide.

Bead Deposition 4. Deposit 3 modified beads onto a glass slide (Figure 3). During bead loading, deposition chambers enable you to segment a slide into one, four, or eight sections. A key advantage of the system is the ability to accommodate increasing densities of beads per slide, resulting in a higher level of throughput from the same system.

Sequencing by Ligation 5. Primers hybridize to the P1 adapter sequence on the templated beads (Figure 4). 6. A set of four fluorescently labeled di-base probes compete for ligation to the sequencing primer. Specificity of the di-base probe

is achieved by interrogating every 1st and 2nd base in each ligation reaction. 7. Multiple cycles of ligation, detection and cleavage are performed with the number of cycles determining the eventual read length. 8. Following a series of ligation cycles, 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.

Primer Reset

9. Five rounds of primer reset are completed for each sequence tag (Figure 5). Through the primer reset process, virtually every base is interrogated in two independent ligation reactions by two different primers. For example, 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). This dual interrogation is fundamental to the unmatched accuracy characterized by the SOLiD System.

Exact Call Chemistry 10.Up to 99.99% accuracy is achieved with the Exact Call Chemistry Module by sequencing with an additional primer using a multi-base encoding scheme.

THIRD GENERATION:

Ion Torrent

The Ion Torrent Personal Genome Machine (PGM) pairs natural sequencing chemistry with semiconductor technology resulting in a revolutionary sequencing process without fluorescence, enzymatic cascades, or optics. When a polymerase incorporates a nucleotide into a strand of DNA, a hydrogen ion is released resulting in a pH change. 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.

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 1.8 5g total DNA required DNA sample should be suspended in 130L TE

RNA Sample Requirements-

RNA should not be degraded RNA should have an RNA integrity number (RIN) greater than 7. 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. Customers will be asked for more DNA/RNA if their sample fails either of the following check points:

Pico/RiboGreen Assay to verify the amount of starting material Bioanalyzer DNA/RNAchip to check the quality of DNA/RNA, ensuring that there is minimal degradation.

Sample Submission: Please contact Christine Schirmer chrissch@email.arizona.edu prior to sample submission. University customers: We are located in the Keating Building, room 124. Please contact Christine Schirmer to arrange for sample drop-off. External customers: Shipping Guidelines-

Samples should be shipped in screw-cap or snap-cap centrifuge tubes secured with Parafilm. Samples should be placed in a box or a larger tube padded with Kimwipes. Samples should be shipped pre-frozen and on dry ice (regular ice packs are okay for overnight shipping of DNA ONLY). We do not have the availability to receive packages on Saturdays or Sundays. Please email tracking number.

Shipping address: University of Arizona Genetics Core Attn: Ion Torrent Sequencing 1657 E. Helen Street Keating Building, Rm. 124 Tucson, AZ 85721 Expected Results:

314 Chip: 10Mb output, 100bp average length 316 Chip: 100Mbps output, 100bp average length Accuracy: >99.99% consensus and >99.6% raw.

Data Analysis: Sequencing service includes raw sequence files, and basic first pass assembly or mapping using Roche 454 software.

http://uagc.arl.arizona.edu/index.php/next-gen-sequencing-services/ion-torrent.html