Rubber gasket vs. Molecular Markers

[michaelbarton]

We're just about to do some 454 sequencing and de novo assembly of bacterial genomes. We're considering using molecular markers to sequence multiple different genomes instead of the rubber gasket to divide the plate because using the gasket reduces the number of reads dramatically.

I understand that using the molecular markers may introduce bias in the sequencing but I don't fully understand this. What are the downsides of using a rubber gasket that we should be aware of?

[flxlex]

If with 'Molecular markers' you mean tagging of your DNA samples, for example using the MIDs available from Roche/454, then the clear advantage is you do not loose wells, as you write.

We experience problems achieving a good 1:1:1 number of reads, even with careful measurement of DNA concentrations and pipetting. I do not know if this is a results of bias as result of using MIDs, but I do not know what really causes it either...

Hope this helps,

flxlex

[Apollo704]

The biggest downside of using the gaskets is - as you mentioned - the loss of available wells (up to ~50% with a 16 region vs 2 region). Another downside with using the higher number of region gaskets, is the increased chance of mis-loading a library/sample swap.
If you are going to pool samples and use molecular markers, we have experienced difficulty getting even representation across all the sample if the pooling takes place prior to emPCR (we attribute this to the normal variability in the emPCR process). Pooling enriched sequencing beads (post emPCR) produces significantly better representation.