UniSeq DNA sequencing frequently asked questions
1. What kind of DNA templates can be used with UniSeq DNA sequencing system?
A: UniSeq has been successfully performed on PCR products up to 5.5 KB and on plasmids up to 6.5 KB. In principle, there is no size limit to the template providing the primer binding site occurs only once in a template (the UniSeq extended S-primer is an 11-mer, so each priming site should only be found once every 4 to the 11 (~4,000,000) base pairs.
2. Is there a difference in the sequence quality or read length obtained using the UniSeq DNA sequencing kit as compared with a normal sequencing reactions?
A: No. Once the S-primer is generated, a UniSeq reaction is essentially identical to a standard DNA sequencing reaction.
3. What is the average success rate of UniSeq DNA sequencing reaction?
A: We have observed in large scale trial with PCR templates an average success rate of greater than 85%. Other trials of UniSeq under less stringent production condition (microbial genome sequencing) with templates of highly variable quality the success rate was approximately 70%.
4. How many UniSeq DNA sequencing reactions has Nucleics performed?
A: We have performed over ten thousand successful DNA sequencing reactions using the UniSeq system.
5. What DNA sequencing chemistries have been used with UniSeq?
A: We have optimized UniSeq for use with the Applied Biosystems BigDye™ vers. 1.1 & 3.1 and Amersham Biosciences DYEnamic™ terminator chemistries. Other chemistries can be used but may require optimization. Please contact Nucleics if you would like to use other sequencing chemistries.
6. How much can UniSeq save me on the cost of a genome sequencing project?
A: This is highly dependent on the level of integration of UniSeq into your project. Simulation studies by Nucleics suggest that saving in excess of 90% can be obtained with a well thought out integration process.
7. Can UniSeq be applied to projects other than genome sequencing?
A: Yes. UniSeq can be a cost effectively technology for other DNA sequencing projects. Examples include full EST sequencing, transposon mutant DNA sequencing, individual BAC clone sequencing. If you think that UniSeq would be helpful for your DNA sequencing application then please contact us and we will advise you if we think UniSeq would be the best option for your needs.
8. How many EO and TO primers are in the UniSeq primer library?
A: There are 256 EO and 512 TO primers in the UniSeq library.
9. Can I use other DNA sequencing diluents or additives with UniSeq?
A: We do not recommend using other diluents or additives other than those provide with the UniSeq kit. nucleics has devoted considerable resources to developing the UniSeq dilutants and additives and the use of other dilutants will result in a reduced sequencing success rate.
10. What is the UniSEQ reaction modifier and reaction enhancer?
A: They are trade secrets so we can’t tell you.
11. What is the shelf life of the UniSeq DNA sequencing system?
A: All components should last for a minimum of 12 months when stored at –20 deg. C.
12. Can I use other volumes for the library than specified in the manual?
A: Nucleics can provide more or less concentrated primer libraries upon request.
13. What operating system does the UniSelect software work on?
A: The UniSelect software has been written in ANSI C and has been ported to x86 Linux, Mac OS X, and Windows 2000 & XP. Binaries for other platforms can be provided upon request.
14. What is the input file format(s) does the UniSelect software accepts?
A: The phred PHD format is the preferred format, however, the UniSelect software can also process files in FASTA format.
15. What file format is the UniSelect output file in?
A: The file is a A tab delimited text file.
16. How long does the UniSelect software take to select a primer pair?
A: Less than a second (benchmarked on Intel Pentium III 800Mhz running Redhat 7.3).
17. What user defined parameters can I change in the UniSelect software?
A: The UniSelect software allows the user to control a wide range of parameters including:
- The input file(s) and directory(s)
- The output file(s) and directory(s)
- A user defined RegEx file parsing
- Directory recursion
- Primer free energy thresholds
- DNA template secondary structure thresholds
- DNA template quality thresholds
- The extent of read overlap
For further details please see the UniSelect software users manual.
18. Can I use the UniSelect output with my pipetting robot’s control software?
A: Yes. The tab delimited text file can be parsed to provide “cherry picking” instructions for most robotic systems (basically any platform can be used that can read tab delimited files). In addition, Nucleics has developed scripts for controlling the Biomek 2000 robotic system. Nucleics will work with your scientists to integrate UniSelect into other robotic systems upon request.