Whole-genome sequence of an Aspergillus parasiticus strain isolated from Kenyan soil

ABSTRACT Aspergillus parasiticus is an important aflatoxigenic fungus, frequently found in soil samples. Here, we report the sequencing of A. parasiticus strain MRI410 using Illumina MiSeq and Oxford Nanopore platforms. This strain was isolated from soil of a Kenyan maize field.

T he filamentous fungus Aspergillus parasiticus is, in addition to A. flavus, the most serious aflatoxigenic Aspergillus section Flavi species. It is described to prefer ground crop hosts to plant hosts and was already repeatedly isolated from peanuts (1,2). To our knowledge, genomes of A. parasiticus have only been sequenced by short-read sequencing technologies until now. To provide a genome assembly of an A. parasiti cus strain sequenced also with a long-read sequencing technology, strain MRI410 was sequenced using Oxford Nanopore in addition to Illumina MiSeq technology.
For the isolation of this strain, soil samples from the rhizosphere of a maize field in Makueni (1.382 S, 37.322 E), Kenya, were diluted using Tween-80/NaCl-mixture (NaCl 9 g/L, Tween-80 1 g/L, and agar 1 g/L), and serial dilutions were incubated on selective nutrient media. Then, individual colonies were separated. The strain was identified by partial sequencing of β-tubulin, calmodulin, and nitrate reductase genes (Fig. 1), and the identification was verified by the Westerdijk Fungal Biodiversity Institute (Utrecht, Netherlands). After the incubation of A. parasiticus MRI410 on malt extract agar (malt extract 17 g/L, glucose 5 g/L, and agar 16 g/L) at 25°C for 4 d and its homogenization in liquid nitrogen, DNA was isolated using the NucleoSpin Plant II kit (Macherey-Nagel, Düren, Germany) following the official protocol. DNA quality and concentration were verified using a NanoDrop 1000 spectrophotometer and a Qubit 3.0 fluorometer (both Thermo Fisher Scientific GmbH, Bremen, Germany). The same DNA sample was used for the following sequencing with Oxford Nanopore and Illumina MiSeq technologies.
For long-read sequencing, a library was prepared using the Rapid Sequencing kit (Oxford Nanopore Technologies, Oxford, United Kingdom). Sequencing was performed on a MinION Mk1C instrument (Oxford Nanopore Technologies, Oxford, United Kingdom) resulting in 573,070 raw reads (1.56 Gb, N 50 6.35 kb). Subsequent base calling of Nanopore data was done using Guppy, and quality was checked using pycoQC (13). For short-read sequencing, a library was generated using the DNA Prep kit (Illumina, San Diego, CA, USA), and its quality was controlled with the Experion DNA 1k analysis kit (Bio-Rad, Feldkirchen, Germany). The 2 × 300 bp sequencing was performed on a MiSeq platform (Illumina, San Diego, CA, USA). A total of 26,943,603 raw read pairs were trimmed (Trimmomatic-0.39) based on the quality control (FastQC 0.11.3) (14).
In total, 55 gene clusters coding secondary metabolites were predicted using antiSMASH 6.1.0 with the cluster finder algorithm for BGC border prediction (22,23). The aflatoxin gene cluster will be further analyzed and compared to that of other Aspergillus species.

ACKNOWLEDGMENTS
The work described here is part of the project AflaZ, which is being funded by the Federal Office for Agriculture and Food (BLE) under the reference AflaZ 2816PROC11.
We thank Charles Nkonge for providing the soil samples, Christian Roder for the isolation of the strain, Karla Hell for her excellent technical support, and Dr. Andreas Dötsch for the bioinformatic support.