NATpipe: an integrative pipeline for systematical discovery of natural antisense transcripts (NATs) and phase-distributed nat-siRNAs from de novo assembled transcriptomes

Abstract

Nat-siRNAs (small interfering RNAs originated from natural antisense transcripts) are a class of functional small RNA (sRNA) species discovered in both plants and animals. These siRNAs are highly enriched within the annealed regions of the NAT (natural antisense transcript) pairs. To date, great research efforts have been taken for systematical identification of the NATs in various organisms. However, developing a freely available and easy-to-use program for NAT prediction is strongly demanded by researchers. Here, we proposed an integrative pipeline named NATpipe for systematical discovery of NATs from de novo assembled transcriptomes. By utilizing sRNA sequencing data, the pipeline also allowed users to search for phase-distributed nat-siRNAs within the perfectly annealed regions of the NAT pairs. Additionally, more reliable nat-siRNA loci could be identified based on degradome sequencing data. A case study on the non-model plant Dendrobium officinale was performed to illustrate the utility of NATpipe. Finally, we hope that NATpipe would be a useful tool for NAT prediction, nat-siRNA discovery, and related functional studies. NATpipe is available at www.bioinfolab.cn/NATpipe/NATpipe.zip.

Figure 1. Summarized workflow of NATpipe.

(A) Two functional modules “NAT prediction” and “Search for phased sRNAs” were integrated into the pipeline. The first module requires de novo assembled transcripts as the input, and the second module requires small RNA (sRNA) HTS data (at least) and degradome HTS data (would be best if available). The parameters in blue color are adjustable. (B) Illustration of the phase-distributed sRNAs identified within the perfectly annealed regions (>80 bp) of a NAT pair. As an example, eight sRNAs with consistent sequence length were assigned to four phases (four sRNAs on each strand). Each phased sRNA duplex (sRNA1/sRNA8, sRNA2/sRNA7, sRNA3/sRNA6 and sRNA4/sRNA5) possesses 2-nt overhangs at their 3′ ends (dashed boxes just indicate two 2-nt overhangs for example).

Figure 2. Graphic presentation of the exemplified output results of NATpipe.

(A) Degradome signatures mapped to the 5′ ends of the phased nat-siRNAs in Dendrobium officinale are expressed by asterisks (blue, brown, green and orange for root, stem, leaf and flower respectively). The degradome signal intensity is shown in the histogram. A total of 20 phases were identified within the perfectly annealed region (marked by a red box) between the two transcripts comp175659_c0_seq1 (annealed from 2429^th to 2848^th nucleotide) and comp168422_c0_seq11 (from 111^th to 530^th nucleotide) based on small RNA (sRNA) sequencing data. For each phase on a strand of the annealed region, the presence of a nat-siRNA in a specific organ is expressed by a colored bar (blue, brown, green and orange for root, stem, leaf and flower respectively). Based on the sRNA sequencing data, expression levels of the nat-siRNAs are shown in the histograms in (B) (for the siRNAs on comp175659_c0_seq1) and (C) (for the siRNAs on comp168422_c0_seq11). There are two biological replicates of the sRNA sequencing experiments. Please note, the y axes of the three histograms are measured in RPM (reads per million) with exponential increment.