Media a potential source of DNA contamination?

[akjones]

Hi Everyone,

I am going to try to make this long story short and sweet. I have an environmental bacterial isolate that I would like to sequence the genome of using the Illumina MiSeq. It was very difficult to get the isolate growing in pure culture and I struggled for several months trying to increase my biomass and speed up generation time (these are strict anaerobes and somewhat of a pain to work with), and I now have the isolate growing happily in a media that contains .025%(w/v) lysed yeast cell powder. My PI is very concerned that the lysed yeast cells I add to the media will contribute S. cerevisiae DNA to any isolate sample that I send for sequencing. I don't share the concern for a few reasons, one of them being that just about every bacterial genome paper I have read has the organism of interest being cultivated in a media that contains some by-product of another organism with foreign DNA (LB, MOYL, brain-heart infusion broth, etc.) and there was no mention of media DNA contribution interfering with sequencing or subsequent genome assembly. I also did a PCR of just lysed yeast cell powder eluted in ddH20 to the same concentration I use in my growth media (similar concept to a colony PCR) using a 17s primer set and ran the product out in a gel. There were 2 very faint bands at around ~250 and ~100bp (expected product size is 580bp and the positive control band was quite bright). Those results tell me that whatever DNA might be present in the yeast powder does not amplify well and is very fragmented, DNA fragments of ~250 and ~100bp would be excluded in the size-selection step of the sequencing library prep anyway. I also think it is very unlikely that a significant portion of any DNA from the yeast media would make it through the DNA extraction process, since there are two centrifugation steps and a pellet wash step specifically intended to remove residual growth media prior to lysis.

Should I be concerned about yeast DNA from the powder? Has anyone else dealt with a similar concern?

I would appreciate any and all input on this. My advisor is pushing me to try and grow my organism on a very limiting media prior to DNA extraction for sequencing (to exclude "contaminating" DNA) and I am really loathe to do that since it negatively affects both the quality and quantity of my extracted DNA.

Thanks,
~Ana

[cliffbeall]

I wouldn't worry about it too much, just wash your bacterial cells as well as you can before prepping the DNA. Several cerevisiae genomes are known, so you should be able to screen out contamination by BLAST. One thing you may need to remember is to screen against both the yeast nuclear and mitochondrial genomes, they are separate in Genbank.

Its not uncommon to get some contamination in next gen sequencing. Human, E. coli and typical cloning vectors are also worth looking out for.

[mcnelson.phd]

If you're using a MiSeq and your PI is already overly concerned about exogenous DNA, then I should warn you about the issues of carry over contamination, phiX contamination, and index misassignment.

The carryover contamination issue was acknowledged by Illumina in a tech bulletin and results in a small number of reads from a previous run being left in the system and they then get incorporated into the next run. The best way to get around that issue is to do a full maintenance wash before you sequence, and to use completely unique indices between runs.

The phiX contamination is almost unavoidable, but you can easily screen out those reads by mapping against the phiX genome.

The index misassignment issue results from the same issue that causes phiX reads to get included in your demultiplexed sample, and the best way to get around it is to sequence just your sample on the MiSeq run. If you must multiplex, then use dual indexing which will limit the number of reads that have both indices incorrectly assigned to the same ones used for your precious sample.