Systems-Based Analysis of the Sarcocystis neurona Genome Identifies Pathways That Contribute to a Heteroxenous Life Cycle

Blazejewski, Tomasz; Nursimulu, Nirvana; Pszenny, Viviana; Dangoudoubiyam, Sriveny; Namasivayam, Sivaranjani; Chiasson, Melissa A.; Chessman, Kyle; Tonkin, Michelle; Swapna, Lakshmipuram S.; Hung, Stacy S.; Bridgers, Joshua; Ricklefs, Stacy M.; Boulanger, Martin J.; Dubey, Jitender P.; Porcella, Stephen F.; Kissinger, Jessica C.; Howe, Daniel K.; Grigg, Michael E.; Parkinson, John

Culturing of parasites, extraction of DNA/RNA, and sequencing. S. neurona strain SO SN1 was isolated from a southern sea otter (19) and obtained from Patricia Conrad, University of California, Davis, CA. S. neurona parasites were maintained in MA-104 cells as described previously (49) . Genomic DNA was extracted from frozen pellets of S. neurona SO SN1 by proteinase K digestion and subsequence phenol-chloroform extraction. Five libraries were prepared: two Roche 454 Shotgun GS-Titanium libraries prepared in accordance with the Rapid Library Preparation Method Manual (Roche), a Roche 454 8-kb paired-end GS-Titanium Library prepared in accordance with the Paired-End Library Preparation Method Manual with the modification of setting up four circularization reactions to increase the final library yield, an Illumina 2-to 3-kb mate pair library synthesized with the TruSeq DNA sample prep kit (Illumina) and run on an Illumina GA IIx, and a Nextera 8-to 15-kb mate pair library prepared in accordance with the manufacturer's recommendations and run on an Illumina HiSeq 2000. These sequencing efforts generated 7,020,033 shotgun reads, 5,919,255 shotgun reads, 1,100,788 paired-end reads, 128,614,194 mate pair reads, and 136,301,151 mate pair reads, respectively. S. neurona SO SN1 RNA was isolated from merozoites with the RNeasy minikit (Qiagen), snap-frozen, and stored at Ϫ80°C. A single TruSeq v2 RNA library (mRNA enriched) was prepared for Illumina sequencing by the standard Illumina protocol and used to generate 59,622,019 reads. For further details of genome assembly and annotation, as well as bioinformatics and experimental analyses, see Text S1 in the supplemental material.