Computational protocol: Species From Feces: Order-Wide Identification of Chiroptera From Guano and Other Non-Invasive Genetic Samples

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Protocol publication

[…] We downloaded all COI sequences representing Chiroptera, Arachnida, and Insecta (Taxonomy search terms) from BOLD [] in April of 2014. Sequences containing ambiguous characters and sequences shorter than 500 nt or longer than 700 nt were removed from the dataset using BioPython scripts []. Sequences were then clustered (90% identity for Arachnida and Insecta, 99% identity for Chiroptera) with USEARCH []. The resulting dataset () was used for primer design and evaluation with PrimerProspector []. We selected for lab testing four forward and three reverse primers (; SFF_145f, SFF_210f, SFF_348f, SFF_351f, SFF_348r, SFF_351r, SFF_492r) designed to produce amplicons of appropriate length.We also evaluated previously developed DNA mini-barcode primers suggested for forensic barcoding of bats (VF1: []; BC1R, BC2F, BC2R, BC3F, BC3R, BC4F, BC4R, BC5F, BC5R, BC6F []; VR1: []). We used PrimerProspector to predict the primer coverage of Chiroptera and lab-tested the primers with a panel of fecal DNA sources. [...] Samples comprised various tissue types and feces from 54 bat species of the families Molossidae, Mormoopidae, Noctilionidae, Phyllostomidae, Pteropodidae, Rhinolophidae, Rhinopomatidae, and Vespertilionidae (). We first tested primers on 100 tissue samples (internal tissues, blood, wing punches, buccal swabs, hair and associated cells, wing swabs used for Pseudogymnoascus destructans [Pd] detection) of 31 bat species to determine whether primers successfully PCR-amplified with various tissue types, whether the products differed between species, and to identify the best performing primer pair combinations. DNA extractions were performed with a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) using the Animal Tissue Spin-Column protocol; tissues were subjected to lysis for at least 12 hours. A pre-extraction step was added for buccal swabs in order to remove the salts of RNAlater solution: we centrifuged samples for 10 minutes at 10,000 x g to pellet cells, replaced RNAlater with 500 μL 1X Tris-EDTA (TE), vortexed, and let swabs soak for 1 hour. We repeated the centrifugation step and removed 1X TE before continuing with the Qiagen Blood and Tissue protocol described above.We then tested primer combinations that exhibited consistently strong amplification and high specificity (single bands on gel) on 86 individual fecal samples of 31 species () to assess performance (specificity to the intended DNA region and taxon despite high levels of background DNA and inhibitors, and amplification reliability) with fecal DNA and to validate fecal-derived DNA mini-barcodes for bats with a priori (visual) species identification. The validation panel comprised feces from various feeding guilds (insectivores, nectarivores, and frugivores) Fecal samples were fresh (collection methods described above), apart from a subset that were held at room temperature for three months before DNA extraction. We subjected feces to the pre-extraction step described above, and performed DNA extraction with a QiaAmp Fast Stool Mini Kit (Qiagen, Valencia, CA, USA) following the human DNA protocol.We PCR-amplified the COI regions using the following protocols. PCRs for low quality and quantity DNA from feces, buccal swabs, and wing swabs contained 2 μL undiluted DNA template in a 10 μL reaction, with 1 μL 10X Mg-free PCR buffer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.4 μM unlabeled primers, and 0.3 U/ μL PlatinumTaq DNA polymerase (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Cycling involved an initial step of 95°C for 10 min, followed by 38 cycles of 60 s at 95°C, 30 s at 60°C, and 30 s at 72°C, and concluding with a final extension step of 72°C for 10 min. PCRs for DNA from internal tissues and wing punches followed the same protocol except DNA was standardized to 2 ng (DNA quantified with NanoDrop 8000 spectrophotometer, Thermo Fisher Scientific), PlatinumTaq polymerase was lowered to 0.05 U/ μL, and the total number of cycles was reduced to 35.We used MJ Research PTC-200 thermocyclers for PCRs, and visualized PCR products on 2% agarose gels. At least one negative control was included in all PCRs for this study. PCR products were purified using the ExoSAP-IT product cleanup protocol (Affymetrix, Santa Clara, CA, USA), and were added undiluted to a sequencing reaction using BigDye Terminator v3.1 kit according to the recommended protocol (Applied Biosystems, Foster City, CA, USA). We then sequenced products in both directions on an ABI3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and edited sequences with Sequencher 5.3 (http://www.genecodes.com), software. Species identity was evaluated using the BOLD identification tool and NCBI’s Basic Local Alignment Search Tool (BLAST; both alignment based), as well as a non-alignment based approach via the program pplacer []. This latter method employs maximum-likelihood and Bayesian phylogenetic placement to assign lab-generated sequences onto a fixed reference tree. We screened Sanger-derived sequences for presence of nuclear pseudogenes (NUMTs) [, ] by assessing trace files for heterozygous peaks and using a function in BOLD that identifies and flags stop codons. […]

Pipeline specifications

Software tools Sequencher, BLASTN, Pplacer
Application Phylogenetics
Organisms Homo sapiens