DNA计算分析（DNA Computational Analysis）

Sequence Analysis .

Automated (Semi-Automated) Sequencers generate a four-color chromatograms on the results of the sequencing gel, as well as a text file of sequence data. The sequencer can not verify the text data it generates. The researcher has to validate sequencing data manually looking at the chromotograms.

In this lab you will analyze the sequence results from your previous lab#3. Also we will design and test primers using a program FastPCR:

1.Process the raw data from sequencer and assemble two readouts into one contiguous DNA sequence.

2.Identify the sequence using BLAST search.

3.Design and verify primers in silico.

The equipment and software:

Laptop

Network connection

Software Staden Package

Software FastPCR

Staden Package

The Staden Package consists of a series of tools for DNA sequence preparation (pregap4), assembly (gap4), editing (gap4) and DNA/protein sequence analysis (spin).

Pregap4 is a tool for pre-processing sequencing chromatogram files (such as those produced by the ABI instruments). It covers sequence format conversions, quality assessment, vector identification, repeat and contaminant screening and interfaces to assemblers.

Gap4 is a program for sequence assembly and editing. It includes a Bayesian consensus algorithm. It uses phred base quality values to calculate the quality of the consensus sequence. This allows the parts of the assembly that require editing or extra data to be easily found, resulting in rapid finishing and a consensus of known accuracy. Gap4 also includes several linked graphical displays that allow the assembly to be viewed at varying levels of detail.

Gap4 stores the data for an assembly project in a gap4 database. Before being entered into the gap4 database the data must be passed through several preassembly steps via pregap4.

1. Copy the base calls from the trace files to text files known as Experiment files. All the subsequent processes operate on the Experiment files.

2. Quality clipping and vector clipping.

Each step is performed by a specific program controlled by the program pregap4. The trace files are not altered, but are kept as archival data so that it is always possible to check the original base calls and traces. Any changes to the data are made to the copy of the sequence in the Experiment file. Gap4 uses the trace files to display the traces, and to compare the edited bases with the original base calls. However gap4 databases do not store trace files: they record only the names of the trace files. This means that if the trace files for a project are not in the same directory/folder as the gap4 database, gap4 needs to be told where they are, otherwise it cannot use them. Ideally, all the trace files for a project should be stored in one directory. The final result from a sequencing project is a consensus sequence and gap4 can write these in Experiment file format, fasta format or staden format. Of course the whole database and all the trace files are also useful for future reference as they allow any queries about the accuracy of the sequence to be answered.

1.Process the raw data from sequencer and assemble t

wo readouts into one contiguous DNA sequence (Staden).

The first step in assembly is to collect the sequence

files that are going to be assembled.

Create a new directory on the C:\ drive.

Download these two files (chrom.T7.zip , class.zip ) and save them in the directory you just created.

Launch Pregap4 from Start menu.

Use Add files button to browse to the directory you created and select the raw sequence files. (highlighted in oval)

Use the Configure Modules tab (highlighted in oval) to switch to configuration options. Check/Uncheck the crosses next to the modules to resemble the picture presented above.

When you select Gap4 shotgun assembly module you are required to supply a name for your new database (highlighted in square) which will be used to store all the information.

Click Run button (highlighted in diamond)