Mate pair libraries with longer read length

[rakesh.ponnala]

Hello All,

We have prepared illumina Mate pair libraries using the standard 2-5 Kb protocol. But one major limitation for illumina mate pair libraries has been there read length. We only can sequence up to 36 bases of a read and can't go higher due to technical difficulties in the protocol (there is every possibility we may end up sequencing in to the other end of the Biotin which ends up giving lots of chimeras). For our analysis, A 36 base read length is not ideal especially if your assembling Homologous Genomes or regions.

Has someone developed a mate pair library protocol where you can sequence up to 100 bases in length without much trouble. I know BGI has one, developed at there facility. But please let me know.. if any one are aware of other than this.

Best,
Rakesh

[HESmith]

You can use the strategy Roche employs for mate pair libraries - insertion of an adapter during the circularization step - to detect the end junction unambiguously. Also, construct larger libraries (400-800bp inserts) at the second fragmentation step, to minimize the likelihood that the 100bp read spans the junction.

[Jon_Keats]

We run the Illumina protocol with 50x50 reads. If you want longer reads on Illumina make the library with the SOLiD library kit and swap in the illumina adaptors. The issue with the illumina prep is that the mate junction is not fixed in the center of the sequenced fragment. So the longer your read the more likely you are to cross the mate junction and end up with a next to useless read pair. To limit this issue you can gel select larger fragments for sequencing after the fragmentation of the mate circles. This way with longer fragments the chance that you cross the junction is less (ie. 50x50 is 100bp reads, with a >400bp insert 25% of the read pairs will cross a junction if the insert is 200bp then 50% cross a junction.

[giulianog]

Can you use the Illumina kit and clone a linker during circularization?

[RCJK]

Attached is an article describing a modified approach, similar to the 454 protocol, but with Illumina adaptors.