CO1 amplicon with blocking primer sequenced for Individual Pig CA13
We collected fecal material as either fresh scat collected from transects within 24 hours of defecation following the methodology of (Kierepka et al. 2016; doi:10.1002/jwmg.21102), or from fecal material taken directly from the colon of culled individuals. Genomic DNA from fecal swabs was extracted using the MoBio PowerSoil-htp 96-well Isolation Kit (Carlsbad, CA). The following primer pair: MICOlintF (5'-GGWACWGGWTGAACWGTWTAYCCYCC-3') (Leray et al. 2013; doi:10.1186/1742-9994-10-34) and PolyShortCoiR (5'-CCNCCTCCNGCWGGRTCRAARAA-3') (Carr et al. 2011; doi:10.1371/journal.pone.0022232) resulted in amplicons of ~200-300 bases and were considered universally optimal for the target taxa of interest. Amplicon DNA yields from each PCR reaction were then quantified using Nanodrop 2000 (Thermo Fisher Scientific Inc.) and Quanticus Fluorometer (Promega).  All PCR reactions were normalized to equimolar concentrations and pooled together before purification using the MoBio UltraClean PCR Clean-Up protocol. Sequencing libraries for each sample were generated in accordance with the Illumina metagenomic sequencing library preparation protocol.  Sequencing was performed on an Illumina MiSeq at the University of Texas Medical Branch Bioinformatics and Genomics Laboratory. Single 501 bp forward reads were generated for the sequencing run. Each individual pig was sequenced twice, once with COI blocking primer and once without. Dual priming oligomers (DPO) were developed using the approach of Vestheim & Jarman (2008; doi:10.1186/1742-9994-5-12) to block host sequence amplification while minimizing blocking interference with other metazoan sequences. The DPO overlapped with the 3' end of the forward universal primer extending into Sus scrofa specific sequence and was modified with a C3 spacer at the 3'-end, which produced the following blocking sequence: 5'-ACCCACCTTTAGCTGGAAACTTAGCCCATGCAGGAGCTTCAGTTGATCTAACAAIIIICTCCCTACACCTT-C3-3'. The efficacy of the blocking primer was tested in vitro and was verified to bind to the extracted DNA using both a 10:1 and 1:1 ratio of blocking primers to amplification primers.
SUB3097761
CA13.CO1.withblockingprimer