Shrinkage of Genome Size in a Plant RNA Virus upon Transfer of an Essential Viral Gene into the Host Genome

Tromas, Nicolas; Zwart, Mark P.; Forment, Javier; Elena, Santiago F.

To analyze the diversity of deletions that could occur in NIb cistron during the evolution of TEV in N. tabacum 35S::NIb (first experiment described above), the full genome of each genotype was reverse transcribed using M-MLV RT (Fermentas) and primer R97-101, then PCR amplified using the Ultra High-Fidelity DNA polymerase Phusion (Finnzymes) and primers F73-80 and R97-101 (supplementary table S1, Supplementary Material online). Seven independent RT-PCR replicates were performed for each reaction and then pooled, to limit possible PCR artifacts. These PCR amplicons were sequenced by GenoScreen (Lille, France) with an Illumina HiSeq2000 equipment (Illumina Inc.) using a paired-end (2 Â 100 b) protocol. The reads were already demultiplexed and adapters removed. Data were then analyzed with GSNAP (Wu and Nacu 2010) . GSNAP aligned both singleend and paired-end reads. GSNAP is designed to detect short and long-distance splicing, but in our case, we used it to detect deletions instead of introns. GSNAP was used with its default set of parameters, and results were parsed using custom Perl scripts: 1) we excluded reads that are mapped on 100 consecutive bases on TEV genome; 2) for the remaining sequences, we identified sequences that mapped in two different virus genome regions with a minimum of eight bases in the 5 0 and 3 0 ends; 3) we excluded reads poorly represented (<10) and showing a low CIGAR (concise idiosyncratic gapped alignment report) string diversity (<10) in order to eliminate possible artifacts; 4) we calculated the distance between the two different regions to define deletion size and position; 5) we calculated the frequency of each deletion detected; and 6) finally, we obtained the number of reads for each deletion variant in R1 and R2 and we compared it to the total number of reads for each lineage.