Computational protocol: The gut microbiota of larvae of Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae)

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Protocol publication

[…] The variable region 2 (V2) of the bacterial 16S rRNA gene was amplified with the primers 27 F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 338R (5′-TGCTGCCTCCCGTAGGAGT-3′) [], modified with Adaptor A (CGTATCGCCTCCCTCGCGCCATCAG) and Adaptor B (CTATGCGCCTTGCCAGCCCGCTCAG), separated by the four nucleotides in italics, respectively, for pyrosequencing (Roche). The analysis was performed on DNAs extracted from a set of three larvae sampled in April 2011 (lot A) in the urban area of Palermo, Italy. PCRs for the biological samples and reagent control were carried out in five replicates with 0.6 U Platinum® Taq DNAPolymerase high fidelity (Invitrogen) in 1X PCR buffer, 2 mM MgCl2, 300 nM each primer, 0.24 mM dNTP and 100 ng of DNA in a final volume of 25 μl. Cycling conditions were: 94°C for 5 min, followed by 35 cycles of 94°C for 20 sec, 56°C for 30 sec and 68°C for 40 sec, followed by a final extension at 68°C for 5 min.Equal volumes of the five reaction products were pooled and purified using the QiAquick Gel Extraction Kit (QIAGEN®). A further purification step was carried out using the Agencourt Ampure XP (Beckman Coulter Genomics), in order to obtain the required pyrosequencing-grade purity, that was assessed by loading a sample in a High Sensitivity DNA chip Agilent 2100 Bioanalyser.PCR products were mixed for emulsion PCR at one copy per bead using only ‘A’ beads for unidirectional sequencing. Beads were subjected to sequencing on the Roche 454 GS FLX Titanium platform (Roche, Switzerland). Sequences obtained were directly clustered (no trimming was required) with CD-HIT 454 software [] using three different similarity threshold: 90%, 95%, and 97%. This software was also used to extract representative cluster consensus sequences. After they were filtered and annotated using the Ribosomal Database Project (RDP) classifier software []. Filtering consisted of deleting sequences shorter than 100 bp or containing a number of unknown nucleotides (N) greater than five. Finally, all sequences (clustered plus singletons) were annotated with RDP classifier using default parameters and then parsed to obtain a readable text file in output.The most abundant unique sequence of each OTU cluster (family or, when possible, species) was selected as representative, then aligned by SINA [], mounted in ARB [] and subjected to chimera check (before submission in GenBank) by Pintail v. 1.1 software []. Rarefaction curves were generated from families of clustered OTUs using EcoSim v.1.2d [], separately for each percentage of similarity.The 97% similarity clustered consensus sequences were deposited in Genbank under accession numbers KC896717-KC896758; raw reads were deposited in NCBI Sequence Read Archive with accession number SRR837401 (reference: BioProject PRJNA196888). […]

Pipeline specifications

Software tools cd-hit-454, RDP Classifier
Databases SRA
Application 16S rRNA-seq analysis
Organisms Rhynchophorus ferrugineus
Diseases Salmonella Infections
Chemicals Acetic Acid