3.5. piRNA Ping-pong and Phasing patterns analysis:

In Step 8 reads are checked again with the Cutadapt program [20], and trimmed reads longer than 23nt are aligned to the genome using Bowtie with no mismatch. The genomic location and the number of times of mapped reads are recorded. Using this information, autocorrelation analysis is executed to identify periodic peaks based on a previous script from [37]. For 5'-to-5' phasing analysis, autocorrelation analysis of 5'-to-5' distance on the same genomic strands are carried out [37]. For 3'-to-5' phasing analysis, the autocorrelation analyses of 3'-to-5' distance on the same genomic strands are carried out and Z-score at distance 0 is calculated. For 24-35nt small RNA ping-pong pattern determinations, autocorrelation analysis of 5'-to-5' distance on the opposite genomic strands are carried out and Z-score at distance 10 was calculated, noting Z-scores over 2 as significant. The 18-23nt RNA (siRNA) duplex analysis is similar except that Z-score at 10 distance 21 is calculated for reads 18-23nt long.

Step 8: Phasing and ping-pong patterns analyses scripts within step8_phasing.sh: sRNA_Phasing_pipeline.sh <sample.24_35.trim.fastq.uq.polyn> <species> siRNA_Phasing_pipeline.sh <sample.18_23.trim.fastq.uq.polyn> <species>