Computational protocol: Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer

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Protocol publication

[…] Cowpox, Vaccinia-MVA, and Vaccinia-Lister were grown in Vero (Modified Vaccinia Ankara or MVA) or BHK cells (cowpox, Lister) until the development of CPE (cytopathogenic effect). Cells were then harvested and DNA extracted using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA, USA). Poxvirus amplicons were then generated by using the consensus primers EACP1 (ATGACACGATTGCCAATAC) and EACP2 (CTAGACTTTGTTTTCTG) [,] and HotStarTaq PCR kit (Qiagen). E. coli K12 (Affymetrix, Santa Clara, CA, USA) DNA was amplified using the 16S-specific primers adapted from probeBase [] S-D-Bact-0008-c-S-20-ONT (GCCATCAGATTGTGTTTGTTAGTCGCTAGRGTTYGATYMTGGCTCAG) and S-D-Bact-1391-a-A-17-ONT (GCTTACGGTTCACTACTCACGACGATGGACGGGCGGTGWGTRCA). PCR products were verified via gel electrophoresis then cleaned up with Agencourt AMPure beads (Beckman Coulter, Miami, FL, USA). The amplicons were prepped using genomic preparation kits (SQK-MAP-002) and the methodology from Oxford Nanopore, which is briefly described below and performed as detailed by the nanopore literature from Oxford Nanopore. 1ug of purified amplicon DNA with 50 ng added lambda phage control DNA was end repaired (NEBNext End Repair Module, New England Biolabs, Beverly, MA, USA) and dA-tailed (NEBNext dA-Tailing Module, New England Biolabs). A hairpin adapter was then ligated to the end repaired and dA-tailed amplicons, followed by an overnight incubation with a motor protein (to facilitate entry of the hairpin and complementary DNA strand into the pore). 6ul of the resulting prepared library was diluted in 140ul of MinION™ loading buffer with 4ul MinION™ fuel mix (Oxford Nanopore Technologies, Oxford, UK). The MinION™ flow cells were primed with 300ul MinION™ loading buffer followed by the loading of the sequencing mix. The flow cell was run for 6 hours for each sequencing run.Template reads in FASTA format were extracted using poRe [] from the HDF5 files output by the MinION™ base-calling software. Those reads were aligned against a reference dataset containing the set of microbial genomic regions predicted to be amplified by the primers used for each dataset using BLASR [] with default settings, or LAST [] using the settings described previously for MinION™ data: match score (r) of 1, gap opening penalty (a) of 1, and gap extension penalty (b) of 1 []. Alignment summaries were output by BLASR and LAST and ‘pileup’ files were generated using Samtools []. Read percentage identity was calculated as described previously for LAST output [], and the percentage identity values for BLASR were used as output. R and ggplot2 [] were used to plot the percent identity and alignment span for each read against its known reference, as well as the number of bases aligned to each reference genome in the larger database. A complete set of instructions for that analysis is provided as supplemental information. […]

Pipeline specifications

Software tools BLASR, SAMtools, Ggplot2
Databases probeBase
Application Miscellaneous
Organisms Escherichia coli