Present address: Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan, USA.
Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method
Article first published online: 20 JUN 2012
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd
Special Issue: Microbial Communities - Structure, Behaviour, Evolution
Volume 14, Issue 9, pages 2526–2537, September 2012
How to Cite
Duhaime, M. B., Deng, L., Poulos, B. T. and Sullivan, M. B. (2012), Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method. Environmental Microbiology, 14: 2526–2537. doi: 10.1111/j.1462-2920.2012.02791.x
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- Issue published online: 4 SEP 2012
- Article first published online: 20 JUN 2012
- Received 30 November, 2011; revised 5 April, 2012; accepted 4 May, 2012.
Fig. S1. Comparison of DNA polymerase efficiency and effect of reconditioning PCR on two phage lysates, TUSD #20 and #23. LA-TaKaRa yielded more product and with a broader size range than PFU Turbo HotStart. Original PCR product is diluted 10× for input in reconditioning reaction, and thus results in a 10-fold increase in product. Also, note the enrichment for high molecular weight product following reconditioning.
Fig. S2. %G + C range of virus communities, as seen using various amplification (linker amplification, phi29 multiple displacement amplification, and linker amplified shotgun libraries) and sequencing platforms (454 pyrosequencing, Sanger sequencing). (A) in-house sequence data, (B) and (C) are identifiable by name and available on the CAMERA web portal, including all metadata and publication references.
Fig. S3. The magnitude of difference in %G + C bias between unamplified and amplified treatments was assessed by the magnitude of difference between the integrated area under their %G + C-bias plot curves (Fig. 3). As cycle number increased (and quantity of starting material decreased), there was a slight trend in the deviation of amplified from unamplified treatments.
Fig. S4. Quality score profile of Illumina reads from 20 pooled freshwater cyanophage genomes (L. Deng and M.B. Sullivan, unpubl. data) generated from a linker amplified library. Boxplot generated by Fastx Toolbox.
Fig. S5. Log-linear relationship between PCR cycle numbers and starting DNA, as it is diluted to extinction. Test was performed with dilution of a single phage genome, H105/1.
Table S1. Detailed break-down of linker amplification method, including time and cost estimates from sample to sequence for a typical 20 l aquatic virus sample.
Table S2. Comparison of DNA polymerase sensitivity and specificity.
Table S3. Comparison of sheared DNA size-fractionation techniques.
Table S4. Evaluation of linker amplification efficiency.
Table S5. Increase in DNA as a result of linker amplification, from both environmental and phage lysate DNA preps.
Table S6. Two-tailed paired Student's t-tests comparing the integrated areas between unamplified read depth curve and curve of each amplified treatment.
Table S7. Two-tailed paired Student's t-tests comparing the integrated areas between unamplified %G + C bias curve of unamplified and each amplified treatment for H105/1.
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