TCGA sequencing facility has been established to provide application specific, high throughput and completely automated data generation and analysis services. The facility is based on processes which are run and managed with exclusive emphasis on high throughput generation of good quality sequencing data and automated high end analysis of generated data, essential for modern day sequence based biology research. Additional services like preparation of template DNA, assay design and optimization as well as downstream advanced analysis modules are available to provide researchers with expanded capabilities for conducting advanced level investigations and projects. In house sample processing and tracking is performed by on line software and data outputs are delivered in real time by FTP transmission. A 24x7 Technical Support Helpdesk is available by email and telephone to interact with customers for project planning and customization of services as well as for resolution of problems and technical advice on data.
Assay Design & Optimization
End to end service. Indicate your target sequence or Genebank/EMBL ID and the regions of interest and we will design and optimize the PCR and sequencing assays for processing of samples.
Genomic DNA and RNA Extraction
We can perform extraction of genomic DNA and RNA for PCR.
High Throughput PCR & Plasmid Purification
We have extensive robotics to set up scaled up PCR and sequencing reactions for high throughput data generation.
We provide primer walking services for large templates with fast turnaround times.
PCR Product, Plasmid, BAC, Cosmid and Phagemid Sequencing
We provide sequencing services for large scale sample processing in significantly less turnaround time. We also purify templates for sequencing with guaranteed delivery of results.
Whole Genome, EST, cDNA Library Sequencing
We can generate about 2 mb sequencing data per day for high throughput sequencing projects.
Resequencing & Mutation Discovery
Resequencing for mutation and/or SNP discovery requires advanced data quality features like PHRED score and quality based automated assembly and database search. We provide customized solution for such projects including complete analysis.
Sequence based HLA Typing, Microbial Identification, Multi Locus Strain Typing and Viral Genotyping
We perform sequence based HLA typing for HLA class I and II. For 16S rRNA gene sequencing based microbial identification, both MicroSeq (Applied Biosystems) 500 and Full Gene are available. We also provide Multi Locus Strain Typing based on MLST Web Tool for microbial strain typing
We also provide TA cloning services using a variety of TA cloning vectors. The service can be customized to include primer designing for the gene of interest to the final cloned product with the sequences.
Whole genome sequencing/Resequencing/Transcriptome sequencing
We also provide high throughput parallel sequencing using platforms like Genome Sequencer (454), Genome Analyzer (Illumina). These platforms are extensively used for sequencing whole genomes, resequencing projects and transcriptome sequencing
3730 Applied Biosystems Genetic Analyzer
We have two state of the art 3730 Genetic Analyzers from Applied Biosystems which can generate 2 mb sequencing data per day.
Hydra, Biomek & Multimek Robotic Workstations.
We have multiple robotic workstations to scale up sample processing and reaction set up to high throughput levels. We have Hydra (Robins), Biomek and Multimek (Beckman) for robotic transfers and set ups.
9700 PCR Thermal Cycler
We use multiple 96-well and dual 384-well 9700 PCR Thermal Cyclers, the industry standard tool for high throughput PCR.
Sample preparation guidelines for sequencing
Please grow the cultures for ten hours in LB Broth or any such simple liquid medium with suitable selection. Pellet the cells by centrifuging 1.5 mL cultures in micro centrifuge tubes and discard the supernatants. Seal the tubes with parafilm before sending them by ordinary overnight courier. In case you prefer to We recommend Qiagen plasmid purification kit for miniprep extraction of plasmid DNA for sequencing. However, please note that in that case we are not responsible for the quality and quantity of template for sequencing.
You can send us genomic DNA for PCR and sequencing. We will optimize PCR,
purification of template and sequencing with control DNA sent by you for
processing your samples. Genomic DNA can be sent in lyophilized form by
ordinary overnight courier. You can also send us PCR products with no
visible artifacts in agarose gels for purification and sequencing. We will
purify templates by Exonuclease I and Shrimp Alkaline Phosphatase digestion
prior to sequencing. In case you prefer to send purified PCR products or
have to perform gel purification of your PCR product (we recommend Qiagen
Gel Extraction kit), please note that we are not responsible for the quality
and quantity of template for sequencing
1. How much DNA do I submit? At what concentration?
We ask you to submit per sequencing reaction
For Plasmid DNA:
500 ng DNA or cell pellets from 5 mL liquid culture.
For PCR Product:
For BAC DNA:
1µg or cell pellets
2. How should I submit DNA for sequencing?
8 or fewer samples: Please submit them in 0.2-0.5-mL micro centrifuge tubes sealed with parafilm.
16 or more samples: You may submit them in 8 tube strip tubes or 96-well plates.
All strip caps and plate covers must be securely fastened to prevent evaporation and/or leakage from the tubes.
3. How should I label my tubes?
Please label tubes containing DNA samples with the name of the sample. Likewise, label tubes containing primers with the name of the primer. Importantly, fill in the sample names and primer concentrations in the sample submission form.
4. What is the best way to determine the concentration of my DNA sample?
By agarose gel verification: Verify the concentration of your sample on an agarose gel along with a size standard ladder to get a fairly good idea of the concentration and size of your DNA.
5. How should I prepare and purify my template and plasmid DNA for sequencing?
Plasmid DNA needs to be prepared with alkaline lysis prep and an ion-exchange resin (such as Qiagen kit).
PCR product We recommend Exonuclease 1- Shrimp Alkaline Phosphatase (EXO-SAP) or Qiagen Kit purification.
6. Does TCGA make direct sequencing of large clones like P1, PAC, BAC, COSMID,FOSMID or PHAGE?
Yes. Let us know the size of your insert and we will help you find the most cost-effective way to proceed.
7.How many bases can we expect for large insert clone sequencing?
Generally, if the template is very clean, we can read about 500 bases for one single-stranded sequencing run on an average template. “Non-average” templates include high G/C, high A/T, microsatellite and those with a long mononucleotide stretch.
8. What primers does TCGA supply?
We provide T7, T3, M13F, M13R, SP6 T7 Terminator, T7 Promoter and many others if they are the "universal primer" on a plasmid. For other vector primers please provide a map of the plasmid containing the primer and we will make it for free.
9. How should I submit my custom primers?
Please submit your custom primer at a concentration of 10 µM (10 pm/µl). We require 5 pmole of primer per sequencing reaction.
10. What about my PCR products? Can you sequence any PCR product?
Yes. For us to sequence a PCR product, it must be a single band on a gel (to avoid mis priming by the sequencing primer and causing an unreadable ‘double’ sequence) at the concentrations stated above. Although we have had success using lower concentrations, we cannot guarantee the success of these reactions. Products showing more than one band on a gel can be gel purified to obtain a single product. However, it is very important to verify the concentration of the PCR product on an agarose gel after the purification procedure, as recovery of gel extracted DNA is almost never 100%. Additionally, the PCR product should not have any kind of fluorescent label (labeled primers or dNTPs) on it, as this would directly interfere with the sequencing reaction. Finally, the PCR product needs to be purified before the sequencing reaction in order to remove the excess primer and dNTPs left from the PCR. We prefer to perform this purification procedure (Exo/SAP enzymatic purification) in our lab, as it allows us to use our quality control procedures to insure that the data you receive is the very best.
11. How many basepairs can I expect from one sequencing reaction of a plasmid/PCR product?
Depending on the template and Primer, we can even get up to 800 bases of sequencing data of average Q20 quality. We guarantee a minimum 500 bases of average Q20 sequencing Data
12. Are there some templates that are harder than others to sequence well? If so, what are they, and why?
Although the sequencing chemistry we use is very robust, and has alleviated most of the difficulties inherent in using earlier sequencing chemistries, some templates are reticent to providing good sequence data:
High G/C templates (>70% GC)—These templates may not stay denatured during the cycle sequencing reaction, making it difficult for the polymerase to incorporate dNTPs and dddNTPs into the growing sequence ladder. Compression can also be a problem in High G/C templates. Compression is caused by two DNA fragments of different sizes migrate to the same position in the gel. All fragments after the first bases showing compression generally do so as well, causing a sequence that may start out robustly to die out rapidly.
High AT templates (>70% AT)—Secondary structures can form in these templates, causing poor polymerase binding to the template, leading to poor sequencing reads.
Samples with high salt concentrations
Different salts will inhibit the sequencing reaction at different concentrations. For instance, empirical evidence shows that sequencing reactions will be inhibited at EDTA concentrations of greater than 1 mM, and the length of read in a sequencing reaction will be noticeably reduced at NaCl concentrations of greater than 40 mM.
Repeatedly frozen and thawed or old templates DNA template may become degraded, leading to much shorter sequencing reads and poor quality data.
Sequence s with long stretches of a single nucleotide
Usually, these are polyA sequences, and can be difficult for BigDye Terminator chemistry to overcome. However, we have techniques that will often allow us to overcome these mononucleotide stretches and obtain the sequence following it. Please remember that if any difficulties arise, we will do our very best to get you the data that you need. For the best sequencing results, please handle your samples according to our recommendations, and call us if you have any questions regarding our services. Other anomalies do exist and we will try to keep you informed if any may appear in your samples.
13. What will I be charged if a reaction fails?
This does not happen very often and when it does, we make every effort to determine what went wrong. If our positive controls indicate a processing failure, we will re-sequence the sample at no charge. If it appears the sample is the problem, we will charge to cover our set-up expenses. We will then work with you to assure that subsequent reactions proceed more successfully.
14. How can I obtain TCGA's Sequencing order form?
The order form is available on our website http://www.tcgaresearch.org/ or call and we will e-mail or fax it to you.
15. Do I have to fill out the whole sample sheet?
Yes, you need to fill the submission form.
16. Help I cannot see my chromatogram traces!! Where can I obtain the program to view my chromatogram traces?
To visualize and print the chromatogram traces, please point your favorite web browser to the following URLs to obtain free software for Windows based computers:
17. What if I need to read a .pdf file?
18. Where can I get another copy of this document and more info about TCGA?
For all this and more, please visit our web page. www.tcgaresearch.org