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 validate the draft A3 locus reference sequence, all P. vampyrus WGS SRA were downloaded from NCBI and locally converted into paired FASTQ data sets using the NCBI SRA Toolkit 2.4.3 (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi; last accessed April 3, 2018). Each data set was subjected to a quality control check using the FastQC program 0.11.3 (Babraham Bioinformatics, Cambridge). Paired FASTQ data sets were imported into CLC Genomics Workbench using the Illumina import function and the CLC Genomics Workbench Next Generation Sequencing (NGS) Toolkit was used for all following tasks. All data sets were trimmed on the basis of quality using the default parameters. Data sets with short paired-read distances were assessed for the presence of overlapping pairs and these were merged where appropriate using the default parameters. The P. vampyrus data set included long paired reads with insert sizes of 2-20 kb which are helpful for the resolution of repetitive gene regions. Validation of the P. vampyrus A3 locus was performed by mapping all reads to the reference sequence construct using the following parameters: Linear gap cost, length fraction ¼ 0.9, similarity fraction 0.9, with automatic detection of paired read distances. Other parameters were left in their default settings. Potential gene coding regions were identified by mapping P. alecto A3 cDNA sequences against the locus through a local BLASTn analysis using CLC Genomics Workbench with default settings. Promoter elements were predicted using the online Neural Network Promoter Prediction 2.2 tool (Reese 2001) (http://www.fruitfly.org/seq_tools/promoter.html; last accessed April 3, 2018) using the default parameters. ISRE were predicted using the online PROMO transcription factor binding site prediction tool (Farr e et al. 2003) (http://alggen. lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_ 8.3 ; last accessed April 3, 2018) using the parameters: Species: Mammalia, Factors: Mammalia.