Table 1.
Summary of the species studied in the present work, their origin, tissue sampled, and the gene analyzed.
Table 2.
Primer pairs used for the amplification of the selected barcode genes.
Fig 1.
(i) The 2D Pass reads produced with the MinION are assembled de-novo using the Loman’s method; (ii) the obtained Loman’s consensus sequence is then BLASTed to retrieve the most similar sequence present in the NCBI database; (iii) the best hit is then selected as the new reference to which the initial 2D Pass reads are aligned using LAST; (iv) the frequency of each nucleotide is calculated for every position along the reference sequence and the final ONtoBAR consensus is generated and (v) BLASTed vs the NCBI database.
Table 3.
Impact of storage temperature on sequencing performances.
Table 4.
Impact of environmental conditions on sequencing performances.
Table 5.
MinION sequencing data from experiments involving different sample preparation protocols.
Fig 2.
Nucleotide frequencies and coverage of aligned reads.
The nucleotide frequencies (bars) of the aligned reads were calculated at every position using the Sanger sequence as reference. The minimum value of the ‘correct nucleotide’ frequency, i.e. corresponding to the reference, along the entire sequence was 0.66. The sequence coverage (continuous line) was obtained by counting the number of nucleotides aligned over each reference position. The frequency value of the four nucleotides and the coverage are shown in a region with average complexity and a homopolymer run, the latter showing a clear drop in coverage.
Fig 3.
Nanopore sequencing coverage vs homopolymer runs.
Read coverage (upper line) and homopolymer runs (bars) along the 532-bp Amietophrynus brauni barcode region is shown. The coverage value of MinION reads was calculated as respect to the reference sequence generated by Sanger. The homopolymer runs indicated with a bar correspond to sequences of at least two identical nucleotides.
Table 6.
MinION sequencing data and sequence identification results.
Fig 4.
The field laboratory set-up for conducting MinION sequencing of amphibians in a montane rainforest of Tanzania.
Fig 5.
NCBI BLAST search of the assembled sequence of Arthroleptis xenodactyloides.
Alignment result of the assembled consensus sequence of A. xenodactyloides (Query, 741 bp) with with the highest score hit retrieved from NCBI BLAST, i.e. A. xenodactyloides sequence (FJ151103.1; 2313 bp), Sbjct. The two sequences matched with 97% identity. Homopolymer sites with more than two identical consecutive nucleotides are highlighted in red while mis-matches are marked by an arrow.
Table 7.
Comparison of sequencing data generated with the old and new MinION flowcell and chemistries.