Genome Skimming: A Rapid Approach to Gaining Diverse Biological Insights into Multicellular Pathogens

Nematode Collection and DNA Extraction Nematodes were collected following methods previously described [1]. Mixed-­‐stages of Anguina agrostis were obtained from infected bentgrass seed. Globodera ellingtonae eggs were obtained from cysts reared on potato in field microplots. A population of Xiphinema americanum originally obtained from a vineyard was cultured in soil on sudangrass in a greenhouse. Populations of Pratylenchus neglectus and P. thornei were obtained from wheat. Individuals of each species were surface sterilized in 0.01% streptomycin sulfate and cultured on sterile carrot disks. Pratylenchus penetrans was collected from raspberry in the field, and reared in soil on raspberry in the greenhouse. Nematodes were obtained by either directly releasing from seeds or cysts (Anguina and Globodera) or by decant-­‐sieving from culture media (Pratylenchus and Xiphinema). After nematodes were collected for analysis (see Table 1 for numbers of nematodes collected), samples were homogenized and total DNA was isolated using a Qiagen DNeasy Blood and Tissue kit (Valencia, CA) for Illumina sequencing. DNA Sequencing We chose the Illumina MiSeq system for our genome skimming approach due to its fast run times and long reads. Genomic DNA libraries were prepared using the Illumina TruSeq DNA Sample Preparation Kit (San Diego, CA) following the manufacturer's instructions. DNA molecules ~650-­‐750 bp were gel-­‐excised following adapter ligation. MiSeq sequencing was performed for 301 X 2 cycles (paired-­‐end). The six PPN samples were multiplexed onto a single MiSeq run using barcode-­‐labeled adapters to distinguish each sample. Sequencing was done at