Double indexed librariers

[tvdvalk]

Hi all,

I am preparing libraries from my DNA extracts but have a question about it. I will pool my samples before doing target capture and then amplify the libraries in a single reaction. Unfortunately, PCR can produce chimeras by recombining different templates molecules (‘jumping PCR’).

The double-indexing protocol from Meyer et al. 2012 overcomes this problem, since chimeras can be filtered out after sequencing based on the double indexes. My question is, does every sample needs it's own unique P5 and P7 index, or is it enough to use unique combinations (so using the same index multiple times)?

I have 90 different samples, If I would use 30 unique P5 and 30 unique P7 primers this would give me 900 different combinations of which I use 90. In this case I would be able to 'filter out' (900 - 90) / 900 * 100% = 90% of all chimera fragments, is this correct?

Thanks!

Tom

[Brian Bushnell]

You must never re-use an index if you want to be able to filter all chimeras. But yes, if you re-use indexes but only use a subset of the available combinations, then if you assume random recombination, your math is approximately correct, though the details depend on which specific combinations you choose.