On site DNA barcoding by nanopore sequencing

<div><p>Biodiversity research is becoming increasingly dependent on genomics, which allows the unprecedented digitization and understanding of the planet’s biological heritage. The use of genetic markers <i>i</i>.<i>e</i>. DNA barcoding, has proved to be a powerful tool in species identification. However, full exploitation of this approach is hampered by the high sequencing costs and the absence of equipped facilities in biodiversity-rich countries. In the present work, we developed a portable sequencing laboratory based on the portable DNA sequencer from Oxford Nanopore Technologies, the MinION. Complementary laboratory equipment and reagents were selected to be used in remote and tough environmental conditions. The performance of the MinION sequencer and the portable laboratory was tested for DNA barcoding in a mimicking tropical environment, as well as in a remote rainforest of Tanzania lacking electricity. Despite the relatively high sequencing error-rate of the MinION, the development of a suitable pipeline for data analysis allowed the accurate identification of different species of vertebrates including amphibians, reptiles and mammals. <i>In situ</i> sequencing of a wild frog allowed us to rapidly identify the species captured, thus confirming that effective DNA barcoding in the field is possible. These results open new perspectives for real-time-on-site DNA sequencing thus potentially increasing opportunities for the understanding of biodiversity in areas lacking conventional laboratory facilities.</p></div

Nanopore sequencing coverage <i>vs</i> homopolymer runs.

<p>Read coverage (upper line) and homopolymer runs (bars) along the 532-bp <i>Amietophrynus brauni</i> 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.</p

Forest field laboratory.

<p>The field laboratory set-up for conducting MinION sequencing of amphibians in a montane rainforest of Tanzania.</p

Nucleotide frequencies and coverage of aligned reads.

<p>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>i</i>.<i>e</i>. 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.</p

Comparison of sequencing data generated with the old and new MinION flowcell and chemistries.

<p>Comparison of sequencing data generated with the old and new MinION flowcell and chemistries.</p

Impact of storage temperature on sequencing performances.

<p>Impact of storage temperature on sequencing performances.</p

MinION sequencing data and sequence identification results.

<p>MinION sequencing data and sequence identification results.</p

The “ONtoBAR” pipeline.

<p>(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.</p

Primer pairs used for the amplification of the selected barcode genes.

<p>Primer pairs used for the amplification of the selected barcode genes.</p

Summary of the species studied in the present work, their origin, tissue sampled, and the gene analyzed.

<p>Summary of the species studied in the present work, their origin, tissue sampled, and the gene analyzed.</p