I'm working on some old dry pinned beetles and am trying to figure out a way to sequence their gut content (plant materials, some may feed on more than one type of plants), what is the best way to go about doing this? Would shotgun sequencing or designing mini-barcode for hybrid enrichment work? Any leads would be greatly appreciated!
Sequencing the Intergenic transcribed spacer is an option, several thousand copies in plants, so a good chance, even with low amounts of slightly degraded DNA. I would suggest the ITS1 region (primers ITS1/ITS2, or the ITS2 region (gITS7/ITS4) and sequencing on a MiSeq.
Have fun! Berthold
The answer to your question is really highly depends on the condition of your samples and the aim of your work. There is no universal barcode for plants. If you have dried plant samples in a good condition, you may isolate DNA and use any markers you need (for such kind of material would be better to choose shorter ones). But if you suppose some probability of contamination (I think that DNA from beetles and also fungi would be present in gut content with high probability), you'll need some more specific markers. Maybe the better solution to your material is using cp DNA to avoid contamination by fungi or insect DNA. atpF–atpH spacer, matK gene, rbcL gene, rpoB gene, rpoC1 gene, psbK–psbI spacer, and trnH–psbA spacer used frequently in plant barcodes today (http://www.pnas.org/content/106/31/12794.full). I think it would be a better solution than rDNA spacers because beetles and fungi which often present in gum, have their own ITS sequences and it would be quite difficult to get a good sequence from the material without cloning.
I hope it would be helpful,
All the best
Andrii
I think the best approach might be to do shotgun sequencing (i.e. Illumina) of whatever you extract and then assemble high-copy regions (rDNA, mitochondrial fragments, plastid fragments) - you can then assess more fully the gut contents.
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