Very Large Mate Pair Libraries

[Daytwa]

I am unclear on why the mate pair library protocol requires gap sizes to be under 5 kb. Is this just due to the limits of column purification or reliable fragmentation?

Has anyone ever tried to prep a library using larger gap sizes?

Thanks.

[volks]

probably the circularization step becomes less efficient with larger gap sizes.

[HESmith]

Three addition considerations:
1) the molar concentration of your library decreases as the size increases
2) size selection is less accurate (i.e., the range is broader) as the size increases
3) larger gap sizes will skip smaller contigs, increasing the errors in assembly

[Daytwa]

Thanks for the responses so far.

Any idea why the Solid and Solexa protocols differ in regards to how they open the circularized product (Restriction enzyme vs shearing)?

[volks]

the solid protocol now uses nick translation to allow for reads longer than 2x25bp as permitted by the EcoP15I restriction enzyme.
the advantage compared to (random) shearing is that the internal adapter is more likely to be located in the middle of the product, and is less likely to be sequenced from either end. but i am no expert on the illumina protocol.

[HESmith]

Shearing is random (in theory), while the restriction digest strategy yields a defined read length (25bp) from each end.

[JakobHedegaard]

We have prepared ~10 Kbp mate-pair libraries using Illuminas v2 kit. But with lower diversity than a smaller insert size library.
It would be nice if an adapter sequence could be included between the two ends facilitating the detection of the "break-point" and thereby reads longer than the 36 bp as recommended by Illumina to avoid (or at least minimize...) chimeric sequences.
Cheers,
Jakob

[riham]

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