Illumina GAIIx Sequencing Service

Transcription

1 Illumina GAIIx Sequencing Service As researchers continue to develop novel applications for next generation sequencers, the technology landscape of the industry continues to advance at an unprecedented pace. Our dedicated genomics team can provide you with the technical support you need to ensure your DNA sequencing project is a complete success. DNA sequencing services are conducted on Illumina Genome Analyzer (GA) IIx platforms equipped with Paired End Modules and Cluster Stations. Our facility is equipped to handle most sample preparation protocols along with data assessment and bioinformatic support. In an effort to accommodate the broadest range of sequencing applications, project timelines and project budgets, we currently provide sequencing at two levels of service, each in three frequently desired sequencing formats. Service Levels We currently offer two levels of sequencing service: Priority: Samples are prepared for sequencing in the next available run (currently 1 2 weeks after sample acceptance). Priority service is priced at a 20% premium over our standard service level. Standard: Samples are prepared and placed in the queue for sequencing in the next available run following the placement of priority samples (currently 4 5 weeks after sample acceptance). Standard (list) pricing applies. Sequencing Formats & Run Times We offer three sequencing formats on the GAIIx instrument. Captured inserts (amplicon, genomic fragment, etc.) can be sequenced in Paired End format (2 x 86bp reads) or Single End format (1 x 86bp or 1 x 43bp reads). Paired End sequencing is conducted by sequencing the insert from both directions (5 to 3 then 3 to 5 ), giving a length of 86 nucleotides for each read (usually with a gap in the center). For Single End sequencing, the insert is sequenced in one direction (5 to 3 ) to a length of 86 or 43 nucleotides. The duration of a sequencing run is dictated by the desired read length in base pairs (bp) the longer the read length, the longer the GAIIx instrument has to run (more cycles). For processing a standard genomic sample we allocate a day for sample assessment, clustering, post clustering analysis, then one additional day for post sequencing data assessment, basic analysis, and data packaging. Run times are

2 subject to change, with other applications (such as Chip seq ) requiring more time for sample preparation. Our available sequencing formats and their run times are summarized in the table below. Single End Format Paired End Format Read Length 1 x 43bp 1 x 86bp 2 x 86bp Sample Prep* 1 Day 1 Day 1 Day GAIIx Sequencing 3 Days 6 Days 12 Days Data Analysis** 1 Day 1 Day 1 Day Total Run Time 5 Days 8 Days 14 Days * Includes qpcr, sample clustering, post clustering analysis ** Includes basic data analysis & packaging Run times are subject to change GAIIx Performance Parameters Read Length Run Time (Days) Output (Gb) 1 x 35bp ~ x 50bp ~ x 75bp ~ x 100bp ~ x 150bp ~ Sequencing output generated using TruSeq SBS V5 kit with PhiX library and cluster densities between 508, ,000 clusters/mm 2 that pass filtering on GAIIx Throughput Up to 6.5 Gb per day for a 2 x 100bp run Reads Up to 320 million clusters passing filter and up to 640 million Paired End reads Performance The GAIIx generates a significant higher yield of bases than Q30 Greater than 90% bases higher than Q30 at 2 x 50bp Greater than 85% bases higher than Q30 at 2 x 100bp

3 GAIIx Sequencing Workflow We have implemented a series of quality control checks for both the samples and the clustered Illumina slide to help ensure that every sequence run is successful and the sequence data generated is of the highest quality. The workflow below represents a typical sequencing run. 1. Completion of Sample Information Form. Sample information is provided on an editable PDF form. Data entered in this form is automatically converted to an.xml file, arrives at our server, and is uploaded directly to our database. This eliminates data entry errors and serves to streamline the sample submission process. Samples are then tracked internally by customer number and order number. The Sample Submission Form is available for download at our website 2. Sample Submission & Storage. For adaptor ligated samples ready for Illumina sequencing, we require a minimum of 10 nmol stock in a final volume of 10 µl of nucleasefree water (more = better). For samples that require fragmentation and adaptor preparation, we require 5 µg of lyophilized, intact genomic DNA. Samples should be shipped overnight at 4 C (ice packs). Upon arrival, samples will be inventoried and stored at 4 C (short term) or 20 C (long term). This information is summarized in the table below. Adaptor Ligated DNA Intact Genomic DNA Quantity 10 nmol 5 µg OD 260/ Volume 10ul (nuclease free water) lyophilized Shipping Conditions 4 C (ice packs) 4 C (ice packs) 3. Accurate DNA Sample Quantification. The density of the bridge amplified DNA clusters on the Illumina flowcell is highly sensitive to the initial DNA template concentration (optimal template amount ~ 6 pmol). Loading DNA template in excess will generate overlapping DNA clusters, resulting in these clusters having a low pass filter (i.e. low confidence), as the cluster finding software will be unable to discriminate individual DNA clusters from one another. In contrast, loading the chip with a suboptimal concentration of DNA template will generate a sparse population of DNA clusters that will sequence very well (i.e. high pass filter), but at the expense of fewer sequences per experiment. To assist in generating optimal cluster densities, the DNA concentration for each sample is accurately measured using quantitative PCR (qpcr) prior to loading a sample onto the Illumina chip. All samples are qpcr analyzed in triplicate on a Bio Rad icycler. Standards and enzymes are provided by KAPA Biosystems. 4. Generation of DNA Clusters on the Flowcell. DNA samples are loaded onto the Illumina Cluster Station. The DNA is bridge amplified within the flowcell to generate millions of DNA clusters. Enzymes and reagents are supplied by Illumina. 5. Imaging of DNA Clusters on the Flowcell. After cluster generation, the DNA clusters are stained with Sybr Gold (Invitrogen) and imaged on an epifluorecnce microscope (Zeiss)

4 equipped with a cooled CCD camera. This procedure allows us to quickly identify and address failures encountered in the cluster generation step prior to moving forward on the GAIIx, which saves time and money. 6. Sequencing of the DNA sample. The prepared flowcell is loaded onto the GAIIx Instrument (equipped with a Paired End Module). A typical sequencing run will proceed for several days. The status of the run and the resulting data will be monitored daily to ensure a successful run. 7. Data Analysis. Our bioinformatics team will assess data generated from all GAIIx runs and conduct the appropriate data analysis as requested by the customer. Calculations and alignments are conducted on a local 32 node computer cluster running several Illuminaspecific and open source analysis tools (with specific emphasis on published tools for citation and precedent purposes). The complete Illumina output is available upon request (for those familiar with the post processing information and analysis generated as part of the sequencing run). Provided the software is publicly available for download, additional computational support is available to users already performing analyses with specific programs (and familiar with specific output). Please contact us for more information and software requests. 8. Data Packaging. Sequencing data is provided in FASTA/FASTQ/BCL/QSEQ format. A typical 1 x 36bp run on GAIIx will generate ~14 GB of data. Data can be transferred via network (FTP, HTTP) using compression (.zip,.gz,.7z) tools or delivered on an external hard drive or flash drive (USB 2.0 connectivity). Please contact us about preferred formats and any additional fees for hardware.

5 Terms of Illumina Sequencing Service Payment 50% payment is required PRIOR to clustering, which usually occurs on the day the GA run begins. Assistance with bioinformatics is charged at a rate of $90 per hour beyond the first (free) hour. Sequencing service subject to the following restrictions Users who cancel a scheduled lane will be charged for labor and sample preparation costs up to the time that a cancellation notice is received, and will be charged full run costs if clustering has begun. No guarantee is made regarding the minimum number of usable reads per sample, nor for the usability of sequence data. Due to potential instrument or supplier problems we make no guarantee on turnaround time. AptaMatrix reserves the right to change policies or prices without prior notice; we will not change policies or prices for samples already accepted into the input queue.

6 Sample Submission For adaptor ligated samples that are ready for clustering we require a minimum of 10 nmol stock in a final volume of ~10 µl nuclease free water. For samples that require fragmentation and adaptor ligation, we require 5 µg of lyophilized, intact genomic DNA. Samples should be shipped overnight at 4 C (ice packs). Upon arrival, samples will be inventoried and stored at 4 C (short term) or 20 C (long term). Adaptor Ligated DNA Intact Genomic DNA Quantity 10 nmol 5 µg OD 260/ Volume 10µl (nuclease free water) lyophilized Shipping Conditions 4 C (ice packs) 4 C (ice packs) The Sample Submission Form is available for download at our website To ensure that your experiment(s) are given highest priority, please the sample submission form to James Crill at jcrill@aptamatrix.com. Please print a copy of the Sample Submission Form(s) and include it with your sample shipment. Samples should be clearly labeled (with permanent marking no stickers) with the same information as found in the enclosed Sample Submission Form. Please ship samples to: Attn: Jamie Crill AptaMatrix, Inc. 100 Intrepid Lane, Suite 1 Syracuse, NY Phone: jcrill@aptamatrix.com

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8 SHIPPING All shipments are FOB Syracuse, New York. Goods and services damaged in transit should be noted on the freight bill by the delivering carrier. All claims for goods and services damaged in transit shall be made against the delivering carrier by the Purchaser. ACCEPTANCE OF GOODS AND SERVICES The Purchaser shall inspect the goods, data or services results within thirty (30) days upon delivery. Failure to inspect within thirty (30) days after delivery shall constitute a waiver of the Purchaser s rights of acceptance and shall be equivalent to acceptance of the goods and services. Purchaser agrees to pay all costs of inspection. If upon inspection goods and services are rejected as nonconforming or for any other reason, Purchaser shall notify AptaMatrix of rejection within thirty (30) days of delivery. WARRANTIES There are no warranties beyond the expressed warranty offered with the sale of each particular product. For products where AptaMatrix is acting as a distributor, AptaMatrix will transfer to purchaser any transferrable warranties or indemnities that the manufacturer of the product provides to onset. In such cases where AptaMatrix is acting as a distributor, no additional warranties are given or implied beyond the manufacturer s warranty, if any. In no event shall AptaMatrix be liable for loss of profits or indirect, consequential, incidental, special or other similar damages arising out of any breach of this contract or obligations under this contract, including breach of warranty, negligence, strict liability, or any other legal theory. LIMITATION OF LIABILITY The Purchaser s sole remedy and the limit of AptaMatrix s liability for any loss whatsoever shall not exceed the Purchaser s price of the product(s). The determination of suitability of products to the specific needs of the Purchaser is solely the Purchaser s responsibility. There are no warranties beyond the expressed warranty offered with the sale of each particular product. Except as specifically provided in this document, there are no other warranties express or implied, including but not limited to, any implied warranties of merchantability or fitness for a particular purpose. No information or advice given by AptaMatrix, its agents or employees shall create a warranty or in any way increase the scope of the expressed warranty offered with the sale of each particular product. CUSTOMIZED PRODUCTS AptaMatrix reserves the right to require prepayment on custom orders. Claims by Purchaser of nonconformance to specification must be made to AptaMatrix within ten (10) days of delivery. Failure to inspect and make a claim within ten (10) days of delivery constitutes acceptance of the goods and services. Orders to Purchaser s specifications are executed only on the agreement that the Purchaser shall indemnify AptaMatrix and hold it harmless for any loss, cost or damages of any nature resulting or arising from any infringement of patents or any alleged infringement of patents or infringement of any other intellectual property rights. INDEMNIFICATION

9 Products supplied by AptaMatrix are not designed, intended, or authorized for use as components intended for surgical implant or ingestion into the body or other applications involving life support, or for any application in which the failure of the AptaMatrix supplied product could create or contribute to a situation where personal injury or death may occur. Products supplied by AptaMatrix are not designed, intended, or authorized for use in or with any nuclear installation or activity. Products supplied by AptaMatrix are not designed, intended, or authorized for use in any aeronautical or related application. Should any AptaMatrix supplied product or equipment be used in any application involving surgical implant or ingestion, life support, or where failure of the product could lead to personal injury or death, or should any AptaMatrix supplied product or equipment be used in or with any nuclear installation or activity, or in or with any aeronautical or related application or activity, Purchaser will indemnify AptaMatrix and hold AptaMatrix harmless from any liability or damage whatsoever arising out of the use of the product and/or equipment in such manner. INTERPRETATION This writing is intended by the parties as final expression of their agreement and is intended as a complete and exclusive statement of the terms of their agreement. No course of prior dealing between the parties and no usage of the trade shall be relevant to supplement or explain any term used in these terms and conditions. Acceptance or acquiescence in a course of performance rendered under these terms and conditions shall not be relevant to determine the meaning of these terms and conditions even though the accepting or acquiescing party has knowledge of the performance and opportunity for objection. Whenever a term defined by the Uniform Commercial Code, as adopted in New York, is used in these terms and conditions, the definition contained in the Code is to control. MODIFICATION These terms and conditions can be modified or rescinded only by a writing signed by both the parties or their duly authorized representatives. APPLICABLE LAW These terms and conditions shall be governed by and construed in accordance with the provisions of the Uniform Commercial Code as adopted by the State of New York. Neither the 1980 United Nations Convention on Contracts for the International Sale of Goods and services nor the United Nations Convention on the Limitation Period in the International Sale of Goods and services will apply to this Agreement or any transaction under it.