deletions | additions       diff --git a/Research_Approach_1_3_4__.md b/Research_Approach_1_3_4__.md  index 04281bb..b77877a 100644  --- a/Research_Approach_1_3_4__.md  +++ b/Research_Approach_1_3_4__.md 
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The V9 region of the 18S will be amplified using the 1391f and EukBr primers according to the earth microbiome project protocols.  *currently looking into literature for "best" V4 region primers - Cassie  possibly: AReuk454FWD1 and TAReukREV3 \cite{STOECK_2010}  _Bioinformatic Analysis:_ Samples will be multiplexed and the resulting DNA libraries will be sequenced using Illumina MiSeq 250 bp paired end sequencing at the UCD Genome Center. Preprocessing and quality assessment will be performed as described by \cite{Smith_2014}. Sequences will be analyzed with a modified QIIME, Quantitative Insights Into Microbial Ecology, workflow using UPARSE to pick open reference operational taxonomic units (OTU’s) at 97% and 99% similarity; taxonomy will be assigned using the RDP Classifier with the SILVA rRNA database and Genbank searches. Phylogenetic trees will be constructed by placing representative sequences for each de novo OTU into the SILVA reference tree with RAxML. The resulting OTU matrix will be rarefied and subsequent data analysis performed in R. Alpha diversity will be calculated using a variety of metrics including the Shannon and Simpson indices to determine intrasample diversity. The beta diversity metric, Bray–Curtis dissimilarity, will be calculated for the OTU matrix to determine intersample diversity. PERMANOVA tests will be used to determine the significance of sample type (ex. sediment), location and environmental measures (salinity, temperature, etc.) on microbial eukaryotic community composition. To visualize and assess patterns of microeukayotic community structure, a nonmetric multidimensional scaling (NMDS) ordination of the Bray–Curtis dissimilarities will be created. Mantel tests will be performed to determine if correlations exist between the bacterial community composition from the 16S rRNA results and the microeukaryotic community composition we determine here.