Real time portable genome sequencing for global food security

Sample collection and DNA extraction

In Tanzania, three cassava mosaic disease (CMD) symptomatic cassava leaf samples (Figure 1, Table 1) were collected from the smallholder cassava farmer 1’s field in Bagamoyo. Disease severity was assessed as described by Legg et al.², where 1 is healthy and 5 shows severe symptoms of the disease, including leaf distortion and stunting of the plant. One more asymptomatic leaf sample was collected at MARI, Dar es Salaam. In total, seven CMD symptomatic plants were collected from farmer 2’s farm in Wakiso district in Uganda. Both Tanzanian and Ugandan samples were collected in September 2017. A total of 12 samples from Kenya were collected in February 2018 from various sources (Table 1). High-quality DNA was isolated using the cetyl trimethylammonium bromide method⁹. Each DNA sample (Table 1) was quantified and the purity checked using a NanoDrop 2000c UV–vis Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) was used to check the purity and quantity of DNA for each sample, and results were recorded in Table 1.

Figure 1. Cassava plant infected with cassava mosaic begomoviruses.

Nanopore library preparation and sequencing

In Tanzania and Uganda, the Rapid Barcoding kit SQK-RBK001 and MinION 9.4.1 flow cells (Oxford Nanopore) were used to process genomic DNA extracted using a standard CTAB method⁹. We utilized the Rapid Barcoding kit SQK-RBK004 with 9.4.1 flow cells in Kenya. DNA was diluted to 700 ng as specified in the library protocol. The SQK-RBK001 (Sept 2017) and/or the SQK-RBK004 (Feb 2018) protocols were performed as described by the manufacturer. In Tanzania and Uganda, the MinION was run for 24 h instead of the recommended 48 h, and in Kenya we had a total run time of approximately 17 h due to power interruptions.

Nanopore bioinformatics

In Tanzania and Uganda, Albacore 2.0.2 was used for base calling. In Kenya, Albacore 2.1.10 was used and the scripts were modified to reflect the newest rapid barcoding kit RBK004. Fastq files were imported into Geneious¹⁰ and a local blast database of all known cassava mosaic begomovirus whole genomes were downloaded from GenBank (program set to megablast, maximum hits=1, scoring (match mismatch 1-2, max e-value 1e-1, word size 28, gap cost=linear) and a local blast was performed on each of the reads generated using the Nanopore device. There are other bioinformatics pipelines that are used for Nanopore data, but the focus of our study was to use a simple, efficient analyses pipeline, which was a local blast database and a well-curated reference dataset¹¹. To ensure we did not miss other begomoviruses or pathogens, we confirmed these in-field results by performing a post diagnostic blast of reads on the Nimbus Cloud at the Pawsey Supercomputing Center (Kensington, Australia) with blast 2.2.31 against the full NCBI nucleotide database to confirm results. For reference, the specific database used was {$ blastcmd –db nt/nt –info} Database: Nucleotide collection (nt) 46,853,753 sequences; 170,830,796,758 total bases Date: Feb 20, 2018 5:04 PM. The data were processed into a blast archive using a blast script with the following parameters (Script attached) {$blastn -query "$file" -db /mnt/nucdb/nt/nt -outfmt 11 -culling_limit 10 -out "out.$file.asn" -num_threads 17 } then converted into XML (for loading into Geneious) and HTML for viewing where possible.

Verification of DNA sequencing results

Traditional PCR was used to verify our DNA sequencing results. In Tanzania and Kenya, two primer pairs: EAB 555F/EAB 555F¹² and JSP001/JSP002¹³, which amplify 556 bp and 774 bp, respectively, were used to detect East African CMVs (EACMVs) and African CMVs (ACMVs), respectively. Specifically, high quality DNA were isolated using CTAB method as described by Lodhi et al.⁹. Electrophoresis and spectrophotometric measurements were used to check the quality and quantity of DNA. PCR reactions were carried out in a 25 µl volume, with 12.5 µl (1X final concentration) of One Taq PCR 2X Master Mix (New England Biolabs), One Taq DNA Polymerase in an optimized buffer with 1.5 mM Mg2⁺ and 0.2 mM each dNTPs in 1X final concentration. A total of 0.2 µM (0.5 µl of each) of primer sets were used in the reaction and nuclease-free water were added to 25µl final reaction volume. The template used was less than 500 ng per reaction. PCR was carried out in 2720 Thermal Cycler (Applied Biosystems) with the following programme: 1 cycle of 3 min at 94°C, then 30 cycles at 94°C for 45 s, 56°C for 45 s, 72°C for 1 min, and a final cycle at 72°C for 7 min. Electrophoresis of the PCR product was run on 1% agarose gel stained in ethidium bromide in 1X TAE buffer in a submarine gel electrophoresis unit and visualized using a BioDoc-It 210 Imaging System m-20V Transilluminator (Thomas Scientific).

In Uganda, the presence of ACMV and EACMV in each sample was detected using a pair of specific primers for ACMV, ACMV-AL1/F and ACMV-ARO/R, and primers specific for EACMV-UG2, UV-AL1/F and ACMV-CP/R3¹⁴. Specifically, the presence of ACMV and EACMV in each sample was detected using a pair of specific primers for ACMV, ACMV-AL1/F (5’-GCC GGA ATC CCT AAC ATT ATC -3’) and ACMV-ARO/R (5’-GCT CGT ATG TAT CCT CTA AGG CCT G -3’) and specific for EACMV-UG2, UV-AL1/F (5’-TGT CTT CTG GGA CTT GTG TG -3’) and ACMV-CP/R3 (5’- TGC CTC CTG ATG ATT ATA TGT C-3’) described by Zhou et al.¹⁴. The primers amplify about 1000 bp for ACMV and 1500 bp for EACMV-UG2 of the Coat Protein and AV2 gene sequences of the ACMV and EACMV genomes, respectively. The PCR reaction was set up using GoTaq® Green Master Mix (Promega, Madison USA). Each of the 25 µl PCR mix contained 12.0 µl of 2X GoTaq® Green Master Mix [containing GoTaq® DNA polymerase, 2X Green GoTaq® Reaction Buffer (pH 8.5), 400 µM dATP, 400 µM dGTP, 400 µM dCTP, 400 µM dTTP and 3 mM MgCl₂], 1.0 µl of forward primer, 1.0 µl of reverse primer, 10.0 µl of nuclease-free water (Amressco, Ohio, USA) and 1.0 µl of DNA. PCR was performed using a Biometra professional thermocycler (Biometra, Gottingen, Germany) programmed as follows: 94 °C for 2 min for initial denaturation followed by 30 cycles of 94 °C for 1 min, 60 °C for 1.5 min, 72°C for 2 min and 72°C for 10 min for denaturation, annealing, extension and final extension, respectively. PCR amplicons were separated by electrophoresis in a 1× Tris-acetate-EDTA (TAE) buffer in a 1.2% agarose gel, stained with ethidium bromide (0.1 mg/ml) and visualized using a U:Genius3 (Syngene, Cambridge, UK) gel documentation system.

The image in Figure 1 was captured in Kiromo-Kitonga Bagamoyo, Tanzania by J.N. using a Samsung s8 smartphone.

An earlier version of this article can be found on bioRxiv (DOI: https://doi.org/10.1101/314526).