I am setting up a metagenomics experiment and expect our samples to have low quantities of microbial DNA. We are interested in sequencing blanks as a negative control and to rule out contaminants. Does anyone have experience with this? specifically:
1. How do you proceed with library prep with blanks if there is "nothing" in the blank samples?
2. How do you sequence the blank-sample libraries if there is undetectable DNA?
In our lab, we always have three negative controls per 96 well plate in first and second PCR and cleaning. We only include them in sequencing if we see any bands in negative controls. If negative controls do not have any band, we just avoid them since it could mess the sequencing depth/coverage and quality. It is also very likely that it will create a trouble in further downstream data processing, if the adaptor and primer sequences and not taken good care in quality control and trimming steps.
Please consult the following papers:
http://ccb.jhu.edu/people/salzberg/BME689/Readings/Salter-etal-BMCBiology2014.pdf
Article Optimizing methods and dodging pitfalls in microbiome research
Sorry I did not get your point. I can help you if you specify a bit more. But I try to explain what I understand and what you may be doing wrong.
- For the meta-genomes the sample preparation is the most important step. Do you performing biological filtering ? If you expect low density microorganism in whole meta-genome you may consider sample fractionation depending upon your sample I can suggest you few protocols. If you are using total DNA extraction kits and you are not doing sample fractionation OMG host dna will be most of the total DNA and from your question I also understand you are not doing amplicon sequencing instead you are doing whole genome sequencing. Because amplicon sequencing wont be so problematic with the 16s primers. Proper DNa extraction for sample type is always the highest effector in good quality library preparations.
- Whenever you have performed total DNA extraction you may perform both 16s and 18s PCR and DGGE or Pulse field run so that you can check whether you have good amount of microorganism are in the sample . I was deliberately half the template DNA in PCR. if microorganism is so low ( in human tissue for example) the halved total DNA extra decrease the 16s product of microorganism and I might not use that to library preparation. If you don't get good result in 16s ordinary PCR you wont get larger bacterial contigs in whole genome sequencing of metagenomes.
- From your question I think you also trying to perform deep sequencing. (sequence bias become more aberrant) If you are performing deep sequencing why are you counting contigs or consensus sequences whose coverage less than 10 you can not trust them they are possibly hybrid sequences that you may not observe in reality As similar type of microorganism can be find and the contig might be a mixture of those similar organisms
- Do you think you need a DNA or RNA ? I don't know your final aim but the host blocking primers are really useful for meta-transcriptome. Let me explain it by the case that I have faced. We have meta-genomic rumen samples from an grazing animal and we found alot slug DNA which is irrelevant to our case as our gene of interest is different. While grazing the grasses they can get alot slugs! And also you may get obsolete dna parts (will no longer functional) from whole sequences(there is a bias in meta-genomes you mainly get high copy number sequences and mainly high copy number genes which may not be functional or even they might be structural protein coding regions or obsolete genes). High copy ->high mutation :). I wish we had meta-transcriptome and use eukaryotic mRNA blocking primers so that we can have perfect meta-transcriptome that will give insights of real case.
- I might help you more if I have an idea about your sample
Good luck for your experiments.
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