Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats as Revealed by APOBEC3 Gene Complexity

Hayward, Joshua A; Tachedjian, Mary; Cui, Jie; Cheng, Adam Z; Johnson, Adam; Baker, Michelle L; Harris, Reuben S; Wang, Lin-Fa; Tachedjian, Gilda

To identify the gene scaffolds representing fragments of the A3 gene locus in the P. vampyrus genome, a BLASTn analysis was performed using the A3 gene products amplified in the cDNA analysis as queries. The P. vampyrus genome was downloaded from Ensembl and all BLASTn searches were performed locally using CLC Genomics Workbench with the following parameters: Match score 2, mismatch cost À3, word size ¼ 5, E-values < 1 Â 10 À5 , gap costs of existence 5, extension 2, with no filtering of regions of low complexity. To extend the sequence of each scaffold, 150 nt at both the 5 0 and 3 0 termini was used as the query sequence for a BLASTn search against the P. vampyrus Illumina whole genome sequencing (WGS) project SRA [SRA: SRP047390] using the NCBI BLASTn suite (http://blast.ncbi.nlm.nih.gov/Blast.cgi; last accessed April 3, 2018) with the following parameters: Program ¼ megablast, maximum target sequences ¼ 500, E-values < 1 Â 10 À20 , word size ¼ 28, maximum matches in a query range ¼ 13, match score ¼ 1, mismatch cost ¼ 2, gap costs ¼ linear, with no filtering or masking. All hits with a Differential Evolution of Antiretroviral Restriction Factors in Pteropid Bats . doi:10.1093/molbev/msy048 MBE percentage identity of 97% or higher were downloaded and assembled using the CLC Genomics Workbench Assemble Sequences tool into a contiguous consensus sequence using the following parameters: Minimum aligned read length ¼ 20, alignment stringency ¼high, conflicts ¼ Vote (A, C, G, T). Consensus sequences were aligned with the original query to confirm that the assembly represented a terminally extended match of the original query sequence; they were subsequently used as a new query in an otherwise identical BLASTn search against the same SRA. This process was iteratively repeated until all scaffolds could be extended out far enough that their sequences overlapped with another scaffold. The extended and overlapping scaffolds were joined end to end to create a contiguous reference sequence construct representing the P. vampyrus A3 gene locus.