Sequencing Core
Sanger Sequencing - The ABI 3130xl and 3730xl Platforms
The ABI 3130xl and 3730xl platforms utilize a combination of PCR, proprietary ABI BigDye3.1 chemistry and capillary electrophoresis to provide high quality sequence reads for specific targets. Read lengths vary from 600 to 1000 bases depending on the sample type and quality.
Sequencing Templates & Primers
The CGT accepts PCR products, plasmids, BACs, etc. for sequencing. To receive high-throughput pricing, the correct quantity of sample must be received in 96-well format (see Table 1). Low-throughput pricing will be applied to all other samples. Please provide 10ul of each sample per reaction. Your samples can be sequenced with either facility stock primers (T3, T7, T7term, M13F, M13R, SP6, PCMBF) or custom primers sent with your order. If you are providing primers, please send 10ul of primer per reaction. However, if you are submitting samples for high-throughput sequencing and you are using more then one primer per plate, they must also arrive in 96-well format with a volume of 10ul per reaction and at a concentration of 20ng/ul or 3.0pmol/ul Table 1. Template Quantity
We accept samples in 8-strip format, 96 well format, or in 1.5 ml microcentrifuge tubes. Average turnaround time is 2-4 working days once the samples have been received. Investigators will be charged regardless of outcome. If reactions fail due to an instrument or chemistry failure, the reactions will be re-run free of charge. For small projects, .ab1 files will be zipped and emailed. For larger projects, other arrangements will need to be made. University of Miami researchers will be billed via IDRs. Outside clients will be billed via purchase orders (POs). If you are planning to use our facility regularly, alternative arrangements to facilitate billing and generate less paperwork can be worked out. Please contact nmaswood@med.miami.edu Quantitation is very important and PCR products MUST be cleaned-up and contain only a single discrete band. If the product does not show up as a single band on an agarose gel, it will not work for sequencing unless it is excised and purified from the gel. The lab is located in the BRB at 1501 NW 10th Ave. Miami, FL 33136. Arrangements must be made with lab personnel through HIHGSequecing@med.miami.edu before dropping off samples. Once arrangements have been made, please use the call box at the main entrance to gain access to the building.
For investigators who have already performed PCR and sequencing reactions, we offer a "machine-only" service. Reactions must be purified and arrive in ABI MicroAmp® Fast 96-Well Reaction Plates, 0.1 ml (part # 4346906 or 4346907) accompanied by a CGT sequencing request form as well as a "machine-only samplesheet". Turn around time for "machine-only" is typically next-day service. Primer design and PCR The CGT, under certain circumstances, will also carry out primer design, optimization and PCR. This can be a labor intensive process and it is often difficult to determine the absolute cost and time to completion for a given project. Please email whulme@med.miami.edu for an initial consultation. NEXT-GENERATION SEQUENCING
The Illumina Genome Analyzer (GA) uses proprietary reversible terminator-based sequencing chemistry to sequence, in parallel, millions of randomly fragmented sequences. These randomly fragmented sequences are ligated to tags that bind to a substrate in one of eight lanes that comprise a flow cell. Attached DNA fragments are bridge amplified to create an ultra-high density sequencing flow cell with hundreds of millions of clusters, each containing ~1,000 copies of the same template. These templates are sequenced using a four-color DNA sequencing-by-synthesis technology that employs reversible terminators with removable fluorescent dyes. This base-by-base approach helps to eliminate sequence-context specific errors and allows for the sequencing of homopolymers and repetitive sequences. The CGT uses this high-throughput system to offer a wide range of genetic analysis applications from resequencing to de novo sequencing, transcriptome profiling to the study of regulatory mechanisms. Investigators will want to consider the necessary read length, coverage and paired-end insert size to supports their research. The amount of sequencing generated varies from 1.5 to 3 BILLION bases per lane. To learn more about the Illumina Genome Analyzer system please visit: Applications on the GA Paired-end sequencing Transcriptome Targeted Capture Epigenetic Analysis Multiplexing Whole Genome Sample Requirements For mRNA-seq, the input is total RNA with an Agilent Technologies 2100 Bioanalyzer RNA Integrity Number (RIN) value greater than 8. The amount of total RNA of each sample should be between 1-10ug (but 5-10ug is highly recommended). Suspend the total RNA in 50ul of nuclease-free water in a 1.5 ml RNase-free non-sticky tube. The library created from this sample prep will be enough to process many lanes of sequencing. Alternatively, you may also provide 100ng of poly-A-selected mRNA. ABI (Life Technologies) SOLiD 3 System Coming soon...Inquire for details The SOLiD 3 System utilizes a two base encoding chemistry that provides researchers with accurate next-generation sequencing. The SOLiD 3 System currently generates over 20 gigabases and 400 million tags per run. The combination of multiple slide configurations and sample multiplexing capabilities allows researchers to analyze multiple samples for a variety of applications. The SOLiD 3 System generates 50 base fragments from samples prepared with insert sizes ranging from 600 bp to 10 kbp. This broad range of insert size, combined with the throughput and flexibility of the 2-flow cell configuration, enables precise characterization of structural variation across the genome. |
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