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I would like to sequence genomes from 2 different organisms using Illumina Miseq. one is Staph. aureus of about 2.8Mbp and the other is a virus of about 13Kb. Is it okay to pool this samples after library preparation and run them in one flow cell? What are the pros and cons?
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Sure. You will need to index (tag) the samples so the reads can be separate after the run. -
Yes, I will index the samples, but I was wondering if there is any effect of having different sizes of genomes pooled together. Will the larger be 'sequenced more' than the smaller or will they get equal representation in the final output? Is there a cut-off of or guideline of what kinds of genomes you can pool together?
I am new at this so it may seem like a silly question...Comment
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The number of reads for each library is dependent on loading not the genome size. Pooling libraries with same fragment length distribution should output reads in proportion to loading. If the library (not genome) sizes are different, then with equal loading more of shorter libraries will be sequenced.
You can pool many types of libraries in one run if the sequencing configuration is similar.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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