[MARMAM] New publications on DNA diet analysis methods

Austen Thomas austen.thomas at gmail.com
Thu Oct 24 09:33:39 PDT 2013


http://onlinelibrary.wiley.com/doi/10.1111/mec.12523/abstract

Thomas AC, Jarman SN, Haman KH, Trites AW, Deagle BE (2013) Improving
accuracy of DNA diet estimates using food tissue control materials and an
evaluation of proxies for digestion bias. Molecular Ecology, Early View
article.

Ecologists are increasingly interested in quantifying consumer diets based
on food DNA in dietary samples and high-throughput sequencing of marker
genes. It is tempting to assume that food DNA sequence proportions
recovered from diet samples are representative of consumer's diet
proportions, despite the fact that captive feeding studies do not support
that assumption. Here, we examine the idea of sequencing control materials
of known composition along with dietary samples in order to correct for
technical biases introduced during amplicon sequencing and biological
biases such as variable gene copy number. Using the Ion Torrent PGM©, we
sequenced prey DNA amplified from scats of captive harbour seals (Phoca
vitulina) fed a constant diet including three fish species in known
proportions. Alongside, we sequenced a prey tissue mix matching the seals’
diet to generate tissue correction factors (TCFs). TCFs improved the diet
estimates (based on sequence proportions) for all species and reduced the
average estimate error from 28 ± 15% (uncorrected) to 14 ± 9%
(TCF-corrected). The experimental design also allowed us to infer the
magnitude of prey-specific digestion biases and calculate digestion
correction factors (DCFs). The DCFs were compared with possible proxies for
differential digestion (e.g. fish protein%, fish lipid%) revealing a strong
relationship between the DCFs and percent lipid of the fish prey,
suggesting prey-specific corrections based on lipid content would produce
accurate diet estimates in this study system. These findings demonstrate
the value of parallel sequencing of food tissue mixtures in diet studies
and offer new directions for future research in quantitative DNA diet
analysis.


http://onlinelibrary.wiley.com/doi/10.1111/1755-0998.12103/abstract

Deagle BE, Thomas AC, Shaffer AK, Trites AW, Jarman SN (2013) Quantifying
sequence proportions in a DNA-based diet study using Ion Torrent amplicon
sequencing: which counts count? Molecular ecology resources 13, 620-633.

A goal of many environmental DNA barcoding studies is to infer quantitative
information about relative abundances of different taxa based on sequence
read proportions generated by high-throughput sequencing. However,
potential biases associated with this approach are only beginning to be
examined. We sequenced DNA amplified from faeces (scats) of captive harbour
seals (Phoca vitulina) to investigate whether sequence counts could be used
to quantify the seals’ diet. Seals were fed fish in fixed proportions, a
chordate-specific mitochondrial 16S marker was amplified from scat DNA and
amplicons sequenced using an Ion Torrent PGM™. For a given set of
bioinformatic parameters, there was generally low variability between scat
samples in proportions of prey species sequences recovered. However,
proportions varied substantially depending on sequencing direction, level
of quality filtering (due to differences in sequence quality between
species) and minimum read length considered. Short primer tags used to
identify individual samples also influenced species proportions. In
addition, there were complex interactions between factors; for example, the
effect of quality filtering was influenced by the primer tag and sequencing
direction. Resequencing of a subset of samples revealed some, but not all,
biases were consistent between runs. Less stringent data filtering (based
on quality scores or read length) generally produced more consistent
proportional data, but overall proportions of sequences were very different
than dietary mass proportions, indicating additional technical or
biological biases are present. Our findings highlight that quantitative
interpretations of sequence proportions generated via high-throughput
sequencing will require careful experimental design and thoughtful data
analysis.

-- 
Austen Thomas, MSc, PhD Candidate
University of British Columbia
Marine Mammal Research Unit
Room 247, AERL, 2202 Main Mall
Vancouver, B.C. Canada  V6T 1Z4
Phone: (604) 837-4399,  Fax (604) 822-8180
Email: austen.thomas at gmail.com
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