LongTrace Sequencing Service FAQ

LongTrace DNA Sequencing System FAQ

What do I need to do to use LongTrace 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 LongTrace version using any web browser.

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

There are no external requirements other than .ab1 traces produced by an ABI 3130, 3100, 3730, or 3730xl DNA sequencer. The traces can be LongTrace processed at any time after they have been collected. The Auto LongTrace users manual is now online so that you can see what is involved in running the LongTrace:Box system.

Can I try LongTrace for free?

Yes. The LongTrace processing service provided by the free version is exactly the same as the commercial ftp service. The only limitation is that an individual user can only process 10 traces a day (this is to ensure that everyone can use the service). If you wish to process more traces then please register for the LongTrace FTP service.

How do I use the free LongTrace service?

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

1. Go to the free LongTrace 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 20 to 120 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 .zip suffix).

If you are having trouble with any aspect of the LongTrace server 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 vi sting our free online QualTrace quality control software.

Who is using LongTrace?

The following facilities are public ally using LongTrace in standard production.

• Tufts University DNA Sequencing Core
• Yale DNA Analysis Facility on Science Hill

Can I trust the results?

This is not a simple question as the 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 LongTrace 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 LongTrace processing to be as reliable as that provided by ABI's KB base caller 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 LongTrace.

I only have scf files – can I use LongTrace?

No. The scf file does not contain the raw data needed for the LongTrace reprocessing. If you have a large sequencing project that you would like to reprocess using LongTrace 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 all their original ABI files.

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

No. We only offer LongTrace as a online service or as a full hardware/software LongTrace:Box package. The computation requirements of LongTrace would require the purchase of a dedicated high performance computer so it is actually works out cheaper for you for us to offer a fully integrated system. This approach also allows us to optimize the LongTrace software for a single hardware system thus improving the processing speed.

In practice, the LongTrace:Box system offer all the advantages of installing the LongTrace software locally since it allows you to process your traces locally without uploading to our server and offers full data privacy.

So how does LongTrace work?

The software underlying LongTrace has involved more than 20 man years of work by our scientists here at Nucleics. The algorithms used are trade secrets, but in brief, LongTrace works by re-processes the raw ABI data contained in each trace file. This reprocessing allows the KB basecaller to better base call the trace data, thus increase sing the read length and reducing the error rate.

Why aren't my traces improved by LongTrace?

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

1. Your DNA sequencer is not support. Only ABI 3730 3100 and 3130 sequencers using POP7 are officially supported by LongTrace. If your traces were collected on an ABI 377, 310, 3700 or MegaBACE instrument we recommend you try our PeakTrace basecaller.
2. Your sequencing run condition is not supported. LongTrace is not able to successfully process all run conditions, especially the rapid run modules and certain custom run modules. It is difficult to predict in advance which run modules are support. We recommend trying the free online LongTrace version with a few example traces collected by your particular run condition. If your trace is not improved we also suggest trying the PeakTrace basecaller as it is able to support some run conditions that LongTrace can't.
3. Length of the read. If a read is shorter than what KB calls perfectly then LongTrace processing will not be able improve the base call as it is already as good as it can be.
4. Strength of raw trace signal. If the raw signal is very weak (near to the signal noise level) then LongTrace processing 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. Remember that LongTrace makes good trace better not bad traces good!
5. Failed sequencing reactions. Obviously if the sequencing reaction has failed then LongTrace will not be able to improve the results.
6. Mixed signal caused by two or more templates. KB can sometime give such traces high quality read lengths. While LongTrace could improve these traces, there is little point as the sequences obtained are wrong.

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 LongTrace 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 25 Q20+ bases. We have recently been able to reduce our costs and have passed on the savings. The new prices are:

• 100 LongTrace processings – US$30.00 (30c per trace)
• 500 LongTrace processings – US$130.00 (26c per trace)
• 2000 LongTrace processing – US$480.00 (18c per trace)
• 10 000 LongTrace processings – US$2000.00 (20c per traces)
• 25 000+ LongTrace processings – us for an individual quote.

To purchase LongTrace FTP Processing 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 LongTrace:Box system cost?

The LongTrace:Box system is available as a monthly lease at three trace processing scales. The cost is $800 for the 5000 traces per month system (16 cents a trace), $1100 for the 10,000 traces per month system (11 cents a trace), and $2000 for the 25,000 traces per month system (8 cents a trace). There are additional discounts for long term leases or larger orders.

If you wish to order the LongTrace:Box system please us to discusses your specific requirements and to obtain a price quote.

Do I have to use any particular FTP program?

No. Any ftp program is fine, we recommend that the open source Filezilla FTP program which is free. To access the LongTrace FTP server use 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.

Who has used the LongTrace DNA sequencing services?

While we would love to tell you, our privacy policy prevents us from revealing this information. Try it yourself on your traces for free - after all this is the best test you can make.

Can I process 3130 and 3100 traces?

Yes. The LongTrace service now support ABI 3130 and 3100 traces. Rapid run traces (ie traces collected with run voltages higher than 12kV) are not supported.

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

Log in here using you ftp user account details.

I have an ABI 3700 DNA sequencer – can I use the LongTrace DNA sequencing service?

No, however, you can use the PeakTrace basecalling software which offers equivalent or better trace read lengths with the 3700.

Can I use the LongTrace service with MegaBACE traces?

No. LongTrace only works with traces collected on the ABI 3730 3100 and 3130 series of DNA sequencers. We have recently released a version of our PeakTrace basecalling software for use with the MegaBACE instruments that will provide equivalent trace improvements with the MegaBACE sequencers.