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Optimizing Design for Gut Microbe Identification by Sequencing
The microbiome plays an important role in the physiology of animals across all species. Gut microbes assist with digestion, and the population is dependent on the animal’s diet, genetics, and environmental conditions. Sequencing data from raw environmental samples has provided insight into the taxa that are present in collected samples. Metagenomic studies aim to identify the most complete set of microbes for optimal results. The 16S rDNA gene of prokaryotic organisms is a universal gene that has been used to identify microbes present in a metagenomic studies. Because the gene is universal, PCR primers can be used to amplify multiple taxa in a single reaction, however this technique may introduce PCR-biased artifacts. Recently, raw metagenomic samples have been directly amplified via shotgun sequencing. This allows researchers to avoid any PCR-bias, and allows for maximum microbial population data. The objective of this study is to characterize the range in depth of coverage as affected by replication and extent of multiplexing. Rumen fluid samples were collected on 16 crossbred ewes divergent in residual feed intake which had been fed forage (n=8) or concentrate (n=8) diets. DNA was extracted and genomic libraries were created from each animal. Libraries, multiplexed 4 per lane, were diluted and sequenced using Illumina’s HiSeq 2000. Resulting reads were quality filtered and aligned to two distinct reference databases of 16S rDNA genes. Reads with a ≥97% sequence identity to the 16S rDNA genes in the databases were used to classify taxa present in each sample. The distributions of taxa present were used to calculate the probability that a particular microbial taxa would be represented when varying the extent of multiplexing. For calculation purposes, library preparation costs of $300.00 per sample, and sequencing costs of $800.00 per lane were assumed. When libraries were multiplexed 4 per lane, a total of 349 taxa were represented across the 16 animals. On average, each animal aligned to 37%, or 129 of the total taxa represented. If multiplexing was increased to 8 libraries per lane, the resulting data would be one less taxa represented, and a return of $800.00. Alternatively, libraries multiplexed with 2 libraries per lane, provide a higher representation of total taxa, gaining 10 more taxa than 4 libraries per lane, and an added expense of $800.00.
Keywords: metagenomics, multiplexing, replication