DNA Sequencing Update

Epigenetics: New kit from ABI to study DNA Methylation

The DNA sequencing and fragment analysis applications we support.

Did you know?

We use an Applied Biosystems 16-capillary 3130xl automated sequence analysis system purchased through our NIH-NCRR shared instrumentation grant (S10-RR020977). It has a fast laser and uses the "POP-7®" multi-use, high-resolution, polymer for fast fluorescent data collection. The system provides automated polymer loading, sample loading, detection & analysis of both DNA sequencing and fragment analysis samples. Ancillary equipment for cycle sequencing is formatted for 96-well plates. We can process 3 x 96-well plates per day. At 800 nts per read that gives a maximum throughput of about 106 bases a week.

We can perform data assembly and analysis for large sequencing and fragment analysis (SNP, AFLP, etc). Call or email regarding help with informatics and research database development.

Salts and EDTA can mess up the Polymerase activity. So, performing a final EtOH/water wash of your templates is a good idea. And you should resuspend in water for best results.

Sequencing Services

Cadillac full-service icon The Cadillac™ (A-mode),

A-mode is most appropriate for new clients, clients with few samples, new projects, and troublesome templates. We take your template and use either your custom primer or one of our common primers and give you an edited sequence data file ready for use. Further data processing and analysis (blast, orf prediction, primer design, etc.: just ask) is available and usually free.

Full-service samples have highest priority. It is most appropriate for new projects and troublesome templates. We will troubleshoot difficult to sequence templates until we get the data you need.

Hybrid DNA sequencing Hybrid (B-mode),

B-mode is most appropriate for clients seeking high priority, accurate sequencing, and ready to use sequencing data. It is economical, but does requires some skill with purification and quantification of template DNA. You save some money by quantifying your own template. We add the primer (either one of our "common" primers, on-hand in the lab, or your custom primer) and other reagents for cycle sequencing. Cycles are modified for the type of template: e.g., high G-C content or large templates. We do troubleshoot the sequencing results, and edit your data. Priority right after our Cadillac service.

For each B mode we will take 10µL of your (template + primer + q.s to 20µL with water) mix, add our BigDye/Better Buffer Mix and cycle as appropriate for your sample. We purify the fluorescent extension products, dry the sample down, re-suspend it in Hi-Di Formamide, and place it on our 3130xl. We will send you the edited chromatograph and text file for you to use as you wish.

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C-mode: inexpensive DNA sequencing Scooter (C-mode),

C-mode is most appropriate for those clients who want to save money, experienced clients with proven sample preparation systems, easy to sequence templates, and high-throughput needs. It is very similar to a B-mode: you give us a 2X solution (template + primer + water, in 20 µL final volume) ready to cycle, but you edit your own sequence data, ... so wear your helmet! Further data processing and analysis is available on a fee-for-service basis. Also, any samples requiring extra reagents will not be able to use mode C, this is considered a B-mode.

You will receive sequence data that is ready for editing. You should edit the base calling for each run before assembly or further analysis. Especially note that the ends of sequences are usually of loow quality and should be truncated.

D mode - go by bicycle (cheap) Do it yourself: "Bicycle" (D-mode),

You can really save some money by doing the cycle sequencing reactions yourself.

This mode is appropriate for experienced clients with proven sample preparation systems and high-throughput needs. D-mode is not for samples that require troubleshooting, additional reagents, special cycling, data processing or analysis: use B-mode for those.

Contact the Core Lab for a complete protocol.

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E-mode: Fragment Analysis, aka AFLP AFLP (Fragment Analysis, E-mode),

Amplified fragment length polymorphism (AFLP) analysis is a mapping technique thus uses selective amplification of a subset of restriction-enzyme-digested DNA fragments to generate a set of labelled fragments of varying sizes that is unique to the sample; also known as a "fingerprint". We have installed the GeneMapper software from Applied Biosystems for performing fragment analsysis on our 3130. They are simple to run: you give us the dye-labelled DNA fragments and we give you a printout with the fragment sizes calculated. We can do additional analysis at $30 per hour.AFLP Protocols from Applied Biosystems.
[Reference: Budowle B., et al. 2000. STR allele concordance between different primer sets--A brief summary. Profiles in DNA 3(3):10-11]

Wikipedia describes Amplified fragment length polymorphism PCR, or "AFLP-PCR" (often AFLP), as a tool used in the study of genetics and in the practice of genetic engineering.

AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA.The technique was originally described by Zabeau & Vos in 1993. The procedure of this technique is divided into three steps:

  1. Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments.
  2. Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences.
  3. Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.

Online programs for simulation of AFLP-PCR
ALFIE - BProkaryotes or uploaded sequences
In silico AFLP-PCR for prokaryotes,some eukaryotes or uploaded sequences

A variation on AFLP is TE Display, used to detect transposable element mobility.

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Your Data

Good DSEQ data

We email your data (for each sample, a four-color datafile with quality scores for each position and a textfile of the called bases) as an archived, compressed file. Use StuffitExpander or the unix utility 'unzip' to recover the uncompressed files. The data is also in the Core Lab directory on the MMI Server. Simply log in as a guest, then navigate to your folder in the CoreLab_Public_DSEQ_data directory.

Unincorporated dye terminators, if not removed from a sample after "cycling", prior to sequencing, will show up as "dye blobs". These dye blobs not only obscure data and interfere with base calling, but can lower base-calling quality scores, aka Phred scores. This link provides comparison of various available Sample cleaning kits ( including our own Millipore 96-well purification kit)

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Tools

Sequence Editing

The Netherlands Cancer Institute (Mek&Tosj in the Neefix Lab) and GeoSpiza Inc. have created and made available very cool trace viewers/editors for all operating systems. And they're free!

You should use one of these applications to confirm the automatic basecalling, and edit the data as appropriate.

Alignment, Assembly and Other Analysis

For sequence alignment, we have 'Sequencher' from GeneCodes. We also use the EMBOSS alignment tools.

If there are bioinformatics tools you would like us to make available for your research let us know. We're glad to help. For ongoing informatics needs and custom projects, we offer informatics services.

Scheduling

Samples are run FIFO (first in, first out). Please have your samples in the lab by 2:00 PM for the most efficient turnaround. We can run 2 and even 3 96-well plates a day.

Links, Notes and Methods

The most convenient way to schedule DNA sequencing is the on-line work request. Email the Core Facility for more information.

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