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DNA Sequencing - Instrumentation & Chemistry

Instrumentation

The facility runs an Applied Biosystems 3730S Genetic Analyser fitted with a 50 cm array, that generates read lengths in excess of 1000 bases. The proprietary Kb basecaller used during data analysis, incorporates advanced algorithms that produce accurate sequence basecalls. The file output ( .AB1 ) is Windows format. The sequence data is supplied to the researcher as an electropherogram (trace file) together with a standard text file of the basecalls. The first 800 bases of each electropherogram are colour-printed for easy desk reference and further manual editing . We strongly recommend that all users perform a full manual edit of their sequence data as a standard research procedure.

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Chemistry

The cycle sequencing chemistry currently used by Micromon is the Applied Biosystems PRISM BigDye Terminator Mix - versions 3.1 and 1.1, both incorporating the key enzyme AmpliTaq DNA polymerase, FS. Version 3.1 is the mix recommended for most sequencing applications. We can supply version 3.1 directly to our regular customers.

While our sequence runs generate in excess of 1000 basecalls, up to 900-1000 bases are routinely called with quality values exceeding QV=20 (error probability of 1.0% or less), the majority exceeding QV=50 (error probability of 0.001% or less).

The quality of the basecalls or resolution, is significantly dependent on the purity of the starting DNA template. A reduction in final resolution and signal quality can be caused by the presence of RNA, salt, protein and other contaminating chemicals. This can be eliminated by effective template purification prior to cycle sequencing and thorough reaction clean up, post-reaction. These are both key factors in the generation of long, highly resolved reads.

Many useful user documents can be downloaded from the Applied Biosystems Genetic Analysis web site. The AB 3730 Sequencing Chemistry Guide is an extensive resource covering all aspects of cycle sequencing and has an excellent troubleshooting section.