PeakTrace Basecaller FAQ

Frequently Asked Questions about the PeakTrace basecaller

PeakTrace DNA Sequencing System FAQ

What do I need to do to use PeakTrace FTP?

Nothing more than a ftp program and your ABI traces. Once you are ready to process more traces you will need a credit card or to set up a standing order account. You can access the free PeakTrace version using any web browser.

What DNA sequencers and run conditons are supported?

 
Supported ABI 3730, 3730xl, 3100, 3500 and 3130 Run Conditions
Supported ABI 310 Run Conditions
Supported ABI 3700 Run Conditions
Supported ABI 377 Run Conditions
Supported MegaBACE Run Conditions

If you don't see your run condition on this list please contact us as we should be able to add support.

How is PeakTrace different to other basecaller like phred or KB?

Other basecallers like phred or TraceTuner work by re-basecalling the processed peak data in the trace file. This means that their basecalling accuracy is limited to the quality of the peaks provided by the ABI data collection software. PeakTrace is different in that it works directly on the raw data. PeakTrace reprocesses the raw data before basecalling. This means that we are able to provide a significantly better basecalls with few errors and longer reads.

PeakTrace is similar to KB, but just superior in data processing and basecalling accuracy.

Why are some of my traces returned as .scf files and some as .ab1 files?

This has to do with the differences between input ABI files. The older sequencers (ABI 377 and ABI 310) do not contain all the correct fields to be able to generate a traces file with both base and quality score values. Because of the way ABI has "designed" the .ab1 file format we are not currently able to generate a valid .ab1 file from these older instrument sequencing files (the data we need just doesn't exist in the original files). With these older style traces we output a standard .scf file as do other external basecallers like phred.

This problem is not unique to PeakTrace - as far as we are aware the only other basecaller able to output .ab1 files with base quality scores is ABI's KB basecaller. Unfortunately, the KB basecaller is not available for the older sequencers like the ABI 377 and ABI 3700. We are working on overcoming this limitation so it may be possible to generate .ab1 traces from these traces in the future.

What do I need to use the PeakTrace:Box system?

There are no external requirements other than .ab1 or .abi traces produced by an ABI 377, ABI 3700, ABI 310, ABI 3100, ABI 3130, ABI 3730, ABI 3730xl, ABI 3500 or MegaBACE DNA sequencer. The traces can be PeakTrace basecalled at any time after they have been collected. Since PeakTrace use the raw data signal it can not basecall .scf trace files as these only contain the ABI processed data.

Can I try PeakTrace for free?

Yes. The PeakTrace Basecaller free service is exactly the same as the commercial ftp service. The only limitation is that an individual user can only process 6 traces a day (this is to ensure that everyone can use the service by not overloading our server). If you wish to process more traces then please register for the PeakTrace FTP service or contact us at to puchase a PeakTrace:Box system.

How do I use the free PeakTrace service?

The free version of Longtrace is very simple to use. The steps are as follows:

  1. Go to the free PeakTrace service page.
  2. Use the web form to select your trace files (you can upload 1 or 2 traces at a time).
  3. Press the "Upload Traces" button.
  4. Wait 10 seconds (the time required depends on the number and type of traces as well as the load on the server).
  5. Download the re-processed traces by clicking on the output file link (this will be the name of your file with a .ab1 or .scf suffix).

If you are having trouble with any aspect of the PeakTrace bascaller or web site please contact us by . If your traces are not improved and were collected using an ABI 3730 sequencer then you may wish to find out the cause by visiting our free online QualTrace quality control software.

Can I trust the results?

This is not a simple question as validation of trace processing and base calling software is very complex (for more information on sequencing validation see Ewing et al. (1998) Genome Research 8: 175 - 194). We have extensively validated PeakTrace using the approach of Ewing at al. on a variety of traces of known sequence generated by several large sequencing centers. We have found that PeakTrace basecalling to be as reliable as that provided by ABI's KB basecaller and more reliable than using phred on ABI 3730 data.

So to answer the question, if you are happy with KB then you will be more than happy with PeakTrace.

I only have scf files – can I use PeakTrace?

No. The scf file does not contain the raw data needed for the PeakTrace reprocessing. If you have a large sequencing project that you would like to reprocess using PeakTrace and only have the scf files, we suggest you request the original abi traces from the sequencing facility that generated the data. Most large sequencing centers archive their original ABI files. If they don't suggest that they start doing so.

Can I buy the PeakTrace software to use on my own machine?

No. We only offer PeakTrace as a online service or as a full hardware/software package.

So how does PeakTrace work?

The software underlying LongTrace has involved more than 25 years of work by our scientists here at Nucleics. The algorithms used are trade secrets, but in brief, PeakTrace works by re-processes the raw ABI data contained in each trace file. This allows the basecalling process to be more accurate as well as providing better looking traces

Why aren't my traces improved by PeakTrace?

There are a number of reasons why PeakTrace can not improve a trace file. The major factors affecting PeakTrace basecalling are:

  1. Length of the read. If a read is shorter than what KB calls perfectly then PeakTrace basecalling will not be able improve the basecall as it is as good as it can be.
  2. Strength of raw trace signal. If the raw signal is very weak (near to the signal noise level) then PeakTrace will not be able improve the trace. The signal strength is particularly critical at the far ends of the trace. This is the most common cause we see for the non-improvement of traces. PeakTrace makes good trace better, not failed traces good!
  3. Failed sequencing reactions. Obviously if the sequencing reaction has failed then PeakTrace will not be able to magically make the reaction work.
  4. Mixed signal caused by two or more templates. KB can give such traces high Q20+ read lengths even thought the resulting sequence data is total garbage. While PeakTrace could do the same with these traces there is little point as the sequence would be totally wrong and hence useless.

For advice on how to improving your DNA sequencing reactions please visit the QualTrace quality control demo for a free automatic inspection of your traces, or the DNA sequencing troubleshooting advice pages.

How much does PeakTrace FTP cost?

The FTP service version of LongTrace is sold on a per trace basis. There is no charge to you unless your traces are improved by a minimum of 5 Q20+ bases. The prices for PeakTrace FTP are:

To purchase PeakTrace FTP basecalling units please visit our secure online shop. Please allow 12 - 24 hours for any purchases to be credited to your account.

How much does the PeakTrace:Box system cost?

The PeakTrace:Box system is available as a monthly lease at six trace processing scales. The costs are:

There are additional discounts for long term leases or large orders. If you wish to order the PeakTrace:Box system please us.

Do I have to use any particular FTP program with PeakTrace FTP?

No. Any ftp program is fine. We think that the free open source Filezilla ftp program is ideal. To access the PeakTrace FTP server from a web browser like IE type ftp://nucleics.com and then enter your user name and password. You can upload or download files by dragging and dropping the trace files onto the browser window.

If you are using Mac OS X we recommend the free ftp program Cyberduck.

Can I process ABI 377, 310 or 3700 traces?

Yes. The PeakTrace service now support all ABI instruments although not all run conditions are supported. If you have a run condition that is not supported please us.

How do I find out how many traces I have processed?

Log in here using your ftp user account details.

Can I use the PeakTrace service with MegaBACE traces?

Yes. We have recently added support for the MegaBACE sequencer. This support is still fairly limited. We current only support processing .abd files collected using the ET terminator chemistry. We hope to add support for other chemistry as we receive example traces from customers.

 

PeakTrace:Box

We have released a standalone version of our PeakTrace Basecaller designed for customers who wish to process their traces in-house. Ideal for customers with data privacy concerns.