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lilletine 04-16-2013 02:21 AM

Including virus samples in an exome run?
Hi all,

We have three viruses (1.3 MB in total) that we would like to sequence. They are so small that it would be really expensive to run an emulsion with these alone, so i am wondering whether it is possible to include them in our next exome run.

In an exome run, we barcode and pool 6 exomes (50 MB). The pool is then split on 6 lanes. We would aim for similar coverage for both exomes and viruses, and would therefore add much less of the virus libraries to the resulting ePCR pool.

- Would this be a problem in the downstream sequencing - that three of the barcodes have much lower representation than the remaining 6?

- Colour balance should be taken care of by the exomes, or not?

- The viruses should theoretically only steal 1-2% of the reads from the exomes, but would the pooling compromise the exome sequences in any way?

We are really grateful for any thoughts and/or previous experience!

Zaag 04-16-2013 02:37 AM

Should be ok. We do that sometimes to check if a experiment worked before we sequence a whole lane, never had problems.

JPC 05-01-2013 03:08 AM

We've done something similar with Yeast (spiking it into an E120)

No the low % of the 3 virus barcodes does not cause you trouble
Yes the colour balance should be unaffected
We don't see the pooling of the exomes being affected however we have found it very difficult to get good balance beween the low % samples

good luck

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