Computational protocol: Evidence of Unique and Generalist Microbes in Distantly Related Sympatric Intertidal Marine Sponges (Porifera: Demospongiae)

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[…] Partial 16S rRNA gene was amplified for clone library preparation with the universal forward primer U789F (5′-TAGATACCCSSGTAGTCC-3′) and reverse primer U1068R (5′-CTGACGR CRGCCATGC-3′) targeting the hypervariable region V6 of both bacteria and archaea . Triplicate PCR reactions were performed for each sponge specimens in 50 µl volume, consisting of similar PCR reaction mixture used for culturable bacteria except an increase in primer concentration (5 µl of 10 µm each primer), with previously reported cycling conditions . PCR products were gel purified with PureLink™ Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen).Purified respective 16S rRNA amplicons were pooled and used as DNA insert for clone library construction using pTOP TA V2 vectors in Macrogen Europe. Positive clones were screened, and clones with ∼300 bp insert were sequenced bidirectional using vector primers.Raw sequence data from 384 clones were searched for vector contamination using VecScreen ( The sequences were further validated and trimmed to about 300 bp with a sequence preprocessing pipeline, SeqTrim , and if necessary manually inspected and edited in BioEdit Sequence Editor . The processed sequences were checked for chimeras and other artifacts using Mallard . Chimera removed dataset (n = 349) were used for further analyses and deposited in GenBank (accession numbers KF171537–KF171885). [...] Clone library sequences were aligned using Nearest Alignment Space Termination (NAST) algorithm implemented in Greengenes 16S rRNA gene database . Sequences were assigned and clustered to OTUs at different cutoff values using furthest neighbor method implemented in mothur software package . OTUs defined at a cutoff value of 0.01 (99% similarity) were included in subsequent biodiversity and community structure analyses. [...] Biodiversity indices and the genetic structure for recovered bacterial community from host sponges and seawater (SW) were estimated with various calculators implemented in mothur package . Bacterial richness calculation included observed species richness (Sobs) and expected richness with Chao1 estimator (Schao1). Simpson's diversity indices, Dsimpson and E1/D were used to estimate the bacterial dominance and evenness. Good's coverage estimator was used to determine sampling coverage. Genetic differentiation among and within sponge-associated bacterial community and surrounding water was compared using AMOVA and HOMOVA . Similarities among the symbiont community structure were evaluated with phylogenetic lineage sorting test (P-test; ). Pairwise comparison of bacterial communities among sponge hosts and seawater was estimated using LIBSHUFF . Two dimensional Principal Coordinate Analysis (PCoA) was performed to visually compare the community dissimilarity among the source using weighted Unifrac algorithm with normalization steps . [...] Cultured bacterial 16S rRNA sequences (n = 31) and OTUs from seawater and respective sponge hosts (n = 142) defined at 99% similarity, and its nearest relative obtained from GenBank database using BLAST search in September 2012 were aligned to greengenes reference database . The taxonomic affiliation is further verified with RDP database to minimize the taxonomic misinterpretation. Maximum-likelihood phylogenetic trees were constructed in PhyML with Nearest-Neighbor- Interchange (NNI) heuristic search method. The best fit evolutionary models, GTR+I+G for isolates and TIM+G+I for clone libraries were adopted under Akaike Information Criterion with correction (AICc) implemented in MrAIC ver. 1.4.4 . […]

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