Say I have 8 metagenomic libraries prepared for the HiSeq, each uniquely barcoded. Would there be detectable differences between the two following sequencing strategies?
1) Sequence each library on its own lane
2) pool all libraries equally and sequence this pool 8 times, once on each lane.
Thanks!
It depends of the sequence depth achieved for a given sample and the complexity (diversity and relative abundance) of the genomes present in the sample. Even sequencing a lot obtaining a lot of information i.e. 10.000X of a bacterial genome equivalence (assuming an average of 5 Mb for a bacterial genome, then 50 Gigabases) if the sample has abundant genomic types at 5% of the total community, still you will have a lot of differences even weh repeating the sequencing of exactly the same sample library in the less abundant types as the coverage will not reach them homogeneously. In conclusion, if you have the same amount of sequence information, you will get similar results in the central metabolism reads and contigs of the abundant types in the two approaches, and variations in the less frequent genomes reads as there will be low coverage, on each trial you will get randomly different patches of them, but if you reach a coverage that may be closer to 20X for a less abundant genome, then it could be possible to reconstruct large parts of those genomes. Albertsen, M., Hugenholtz, P., Skarshewski, A., Nielsen, K. L., Tyson, G. W., & Nielsen, P. H. (2013). Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nature biotechnology, 31(6), 533-538. A recent report shows the expansion of the bacterial and archaeal genomic representatives reported for many new groups but any of them was reconstructed from metagenomes, but from single cell genomics or attaining a culture.Mukherjee, S., Seshadri, R., Varghese, N. J., Eloe-Fadrosh, E. A., Meier-Kolthoff, J. P., Göker, M., ... & Yoshikuni, Y. (2017). 1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life. Nature biotechnology.
There is variation in every measurement in biology, but the run-to-run variation in sequencing is often much less than the variation in your 'treatments' you are evaluating (control vs perterubration or across different sample types). So, doing pilot-scale shallow sequencing across many samples and then deeply sequencing select ones is a good strategy.
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