Hi Mariac,

 So many things go into designing/developing a robust enrichment protocol, and it took us years to develop and refine ours. Everything from probe design to post enrichment PCR can play important parts, and improving one without the other may mask the improvements you are looking for. I can't tell you everything I know about improving enrichment (I hope you understand why), but the above suggestions are good places to start. But, the time you will save by using a commercially available kit might outweigh the cost. Good luck!

Best,

Casey

Thank you for your answers.

We tested commercial kits (mybaits from microarray-RNA 80 mers probes) to capture nuclear genes (600kb) and also use home made protocols (DNA probes with a 300 bp mean size) for plastids capture (150kb). Our custom protocol use truncated adapters (34bp) during hybridization to reduce daisy chain but we didn't add blocking oligos, so we 'll test.

Genes enrichment  produced 8-17 % on target reads (40-80 fold enrichment compared to a non enriched libraries). For plastids the on target vary between 20-95 % (similar fold enrichment) depending on species (may be due to differences in nuclear size genome among species) but variations are also observed at intraspecific level.

We work on plants for witch CotI is not available but we could include salmon sperm DNA. How SSD act during in solution capture ?

Could you give some results you obtained in % of on-target reads and enrichment fold, do you think it could be relate to size target / genome size ?

What about the ratio between probes and total DNA library during hybridization ? We actually work with 100 ng probes / 200-500 ng lib. Do you think it can have an effect to the on-target rate ?

Thanks,

Cédric

If your targeted region is relatively small in size you should use a double capture protocol, so repeat the capture hybridisation process ones more. You will see a significant increase on the percentage on target. You can reduce the hybridisation times, in this way the total workflow will be the same concerning time, but labor is doubled.