Oey et al. BMC Genomics 2011, 12:315 http://www.biomedcentral.com/1471-2164/12/315 our analyses differ in that we investigate the genomic origin of most of these 19mers, including those derived from the repetitive IAP and LINE elements, rather than limiting the analyses to non-repetitive RNA. We find that the 19mers are readily distinguished from other RNAs by the composition of their downstream flanking sequence and use this feature to identify likely substrates of piRNA-directed RNA cleavage. Methods Deep sequencing datasets and initial data processing Three publicly available spermatogenic tubule small RNA deep sequencing datasets published by Robine et al [7]. were obtained from the Gene Expression Omni- bus (GEO) [13] under the accession number GSM475279, GSM475280 and GSM475281, and used for the primary analyses. Additionally, small RNA sequencing data from several tissues published by Chiang et al [14]. were also obtained under the GEO Series accession GSE20384 and used for some analyses, as specified in the results. Prior to mapping, all reads containing homopolymers > 8 nt were removed and reads matching the partial sequence CCGGGTTTCGGCACC, identified as tRNA-derived, were also removed. In the case of reads mapping to LINE elements, reads matching the patterns (GGA)4, (CCT)4, (GAA)4 and (CTT)4 were also removed as these were found to cause mapping artefacts against common simple repeats inside many LINE elements. All other data processing and sequence analyses were carried out using scripts available as part of the Biopieces (http:// www.biopieces.org). Mapping of reads to LINE and IAP Reads likely to originate from IAP or LINE elements were identified by mapping the reads to a database con- taining all mouse IAP elements and one containing all LINE elements annotated in the UCSC Genome Brow- ser RepeatMasker track (accessed 01.09.2010) [15]. To obtain reads likely to originate from IAP or LINE repeats the program Bowtie [16] was used to map reads directly to the IAP and LINE sequences using the options “-v 0 -m 50000” to get high-confidence IAP and LINE small RNA datasets. These reads were then remapped to IAP or LINE consensus sequences with less stringent parameters allowing for two mismatches (Bowtie option -v 2). The consensus sequences that were used were obtained from RepBase [17] under the identifiers “IAPLTR1_MM_LTR” and “IAPLTR1_MM” for IAP and “L1_MM” for LINE. The two IAP reference sequences were joined such that the IAPLTR1_MM sequence was flanked on both sides by a copy of the IAPLTR1_Mm_LTR sequence to obtain an IAP refer- ence sequence that includes LTRs. The sequence Page 2 of 10 composition of pr19RNA and piRNA reads were visua- lised in the form of sequence logos that were plotted using the “plot_seqlogo” script, which is available as part of the Biopieces (http://www.biopieces.org). The script calculates logos using Shannons general formula for uncertainty [18,19]. Genome-wide annotation of 19mers All 19mers from GSM475281 were mapped to the mouse genome (NCBI37/mm9) using Bowtie [16] allow- ing reads to map to < 6 loci. Reads mapping to multiple loci were randomly assigned to single loci. The sequence of all the reads plus 10nt of downstream flanking sequence was obtained, and the reads were binned according to the identity of the 10th downstream base. The origin of the reads were then found by intersection against UCSC Genome Browser RepeatMasker annota- tion tracks followed by intersection with piRNA clusters, using 94 piRNA clusters annotated by Lau et al. [20], and finally with RefSeq genes, which were also obtained through the UCSC Genome Browser [15]. Positional relationship of IAP-derived p-piRNAs and s- piRNAs The tendency of p-piRNAs and s-piRNAs to be posi- tionally fixed to pr19RNAs was investigated by first identifying high-confidence pr19RNAs as well as p-piR- NAs and s-piRNAs. The pr19RNAs were defined as reads in same orientation as the IAP reference sequence with the sequence motif N19VN8A, where N19 repre- sents the pr19RNA and V represents any nucleotide except U. The p-piRNAs were reads in the opposite sense of the IAP reference sequence, 24-30 nt in length and with the sequence motif UN8BNn, where B repre- sents any nucleotide except A. The s-piRNAs were any read in the same orientation as the IAP reference sequence, 24-30 nt in length and with the sequence motif VN8ANn, where V represents any nucleotide except U. The 5’ ends of the s-piRNA 5’ ends and p- piRNA 5’ ends were then tallied at incrementally increasing distances from pr19RNAs. The positional relationship between these were elucidated by plotting the density of piRNA 5’ ends relative to pr19RNA 5’ ends, similar to the method employed by Aravin et al. [11]. Distribution of prRNA relative to s-piRNAs across the genome The pr19RNA, s-piRNAs and p-piRNA sequence pat- terns were also used to define regions in the mouse gen- ome enriched for pr19RNA and s-piRNAs by selecting those regions where uniquely mapping pr19RNAs out- number other 19mers, and where uniquely mapping s- piRNAs outnumber p-piRNAs. Regions enriched for