I want to analyse gut of boreal wolf spiders for a whole range of possible prey via Illumina Miseq and targeting the COI gene. suggestions, anyone?
You may find the best approach is to use general primers that will amplify everything and then simply excluded wolf spider sequences. Alternatively, you could use blocking primers to excluded the wolf spider. I guess finding a single primer pair that will amplify all invertebrates and fit for Miseq is no easy task, and you may have to amplify different groups with different pairs separately. This can be quite a job, bringing you back to the two previous suggestions.
I don't know if you've looked at Shepperd et al, maybe something there (EDIT: sorry I think they may have just looked for aphid markers): http://www.ncbi.nlm.nih.gov/pubmed/16313606
If you have access to MiSeq, can you afford to go deep enough to sequence everything and analyse with e.g. MEGAN or MG-RAST, rather than amplifying a specific marker?
Hi,
What about the LCO/HCO and other primers? You can see more info in:
Folmer, et al. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 3(5):294-9.
These are great for invertebrates, but, as Russell said, you may have to block the wolf spider sequences. Hope that helps!
Thank you all very much for the answers- i read some papers meanwhile and i guess i have to use some group-specific primers in parallel for optimal results.
Look into PNA primers to block amplification of the host sequences if the amount of host DNA is significant. http://www.nature.com/nmeth/journal/v10/n10/full/nmeth.2634.html
For those who is also interested in this theme: there's couple of papers where authors analyse the sequence of SSU rRNA gene to find sites universal for all living creatures. Of course you have to evaluate and test primers for your purposes and objects, but these works are of great use as a starting point.
1. Wang Y, Tian RM, Gao ZM, Bougouffa S, Qian P-Y (2014) Optimal Eukaryotic 18S and Universal 16S/18S Ribosomal RNA Primers and Their Application in a Study of Symbiosis.
2. Bradley IM, Pinto AJ, Guest JS. 2016. Design and evaluation of Illumina MiSeq-compatible, 18S rRNA gene-specific primers for improved characterization of mixed phototrophic communities.
As for universal invertebrate primers LCO/HCO they're found to fail with many invertebrate organisms and aren't now considered as universal. See Sharma & Kobayashi 2014.
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