Clip

Human gene annotations were acquired from GENCODE v17 (31 (link)). Protein-coding transcripts were defined as those with ‘protein_coding’ gene biotype and ‘protein_coding’ transcript biotype. The lncRNAs transcripts were defined as those with ‘processed_transcript’, ‘lincRNA’, ‘3prime_overlapping_ncrna’, ‘antisense’, ‘non_coding’, ‘sense_intronic’ or ‘sense_overlapping’ gene biotype. Small non-coding RNA (sncRNA) transcripts were defined as those with ‘snRNA’, ‘snoRNA’, ‘rRNA’, ‘Mt_tRNA’, ‘Mt_rRNA’, ‘misc_RNA’ or ‘miRNA’ gene biotype. Pseudogene transcripts were defined as those with ‘polymorphic_pseudogene’, ‘pseudogene’, ‘IG_C_pseudogene’, ‘IG_J_pseudogene’, ‘IG_V_pseudogene’, ‘TR_V_pseudogene’ or ‘TR_J_pseudogene’ gene biotype.
Mouse and Caenorhabditis elegans gene annotations were extracted from Ensembl Gene Release 72 and LiftOver to mm9/mm10 and ce6/ce10, respectively. Protein-coding, lncRNAs, sncRNAs and pseudogenes were classified using a similar method. Human, mouse and C. elegans circRNA annotations were downloaded from circBase v0.1 (6 (link)).
These transcripts were scanned to find conserved miRNAs target sites using miRanda v3.3a with the ‘-strict’ parameter. The target sites that overlap with any entry of the aforementioned AGO CLIP clusters were considered as the CLIP-supported target sites.

The rich feature set of FLEXBAR addresses many potential applications in single-end, paired-end and mate-pair sequencing. Typical workflows involve a quality-clipping step, demultiplexing, which potentially includes barcode trimming, followed by a separate adapter trimming step. All of these steps may be executed within the same FLEXBAR program call (see Figure S1). The default parameters of FLEXBAR are optimized to deliver good results (especially Illumina and SOLiD) for a large number of scenarios (see benchmarks). However, customization of settings might improve results for specific applications.
FLEXBAR has been implemented in C++ using the Seqan library [1 (link)]. Multi-threading has been implemented with the Intel Threading Building Blocks library [2 ]. FLEXBAR detects target sequences by overlap sequence alignment, based on the Needleman-Wunsch algorithm [3 (link)]. An overlap (or semi-global) alignment uses the same recurrence relations as a global alignment but does not penalize gaps at the end of the alignment (Figure 1A and Figure S2). To this end, the first row and column of the dynamic programming matrix are initialized with zeros and the alignment score maximum is searched in the last row and column of the alignment matrix.
FLEXBAR offers maximal flexibility in target sequence recognition by considering base substitutions, insertions and deletions. Moreover, the user is free to choose all alignment scoring parameters, the minimal overlap and a threshold on sequence similarity. Default parameters are preset for these parameters and were chosen to work best for Illumina HiSeq and ABI SOLiD sequencing data. A simple direct matching to expected sequence tags might not be adequate for sequencing platfoms with elevated indel error rates. Furthermore, letter- as well as color-space encoded reads can be processed (Figure 1A). FLEXBAR supports five sequence trimming modes, which cover most sequencing applications: (1) LEFT, (2) LEFT-TAIL, (3) RIGHT, (4) RIGHT-TAIL or (5) ANY(where) trimming (Figure 1B). These modes can be independently combined for adapter and barcode sequence recognition in single or paired-end data. Barcode reads might be even separated from the actual single or paired-end read set (as in Illumina TruSeq^TM sequencing).

Conserved miRNA families were defined as those labeled with ‘highly conserved’ or ‘conserved’ in TargetScan Release 6.2 (25 (link)). miRNA IDs from miRBase Release 20 were used (26 (link)). Genomic coordinates of these conserved miRNAs target sites predicted by TargetScan (25 (link)), miRanda/mirSVR (27 (link)), PITA (28 (link)), Pictar 2.0 (19 (link)) and RNA22 (29 (link)) were collected and converted to hg19, mm9/mm10 and ce6/ce10 assemblies using LiftOver, respectively. The resulting coordinates were intersected with the previously described Ago CLIP clusters using BEDTools v2.16.2 (30 (link)). The target sites that overlap with any entry of the Ago CLIP clusters were considered as CLIP-supported sites.

The reproductive organs were individually dissected from the newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population, and the relative transcript levels of clip-domain serine protease genes and PPO were examined by RT-qPCR assays. The male reproductive organs were also examined to determine the conversion of PPO to PO in western blot assays using PPO and histone H3 antibodies (0.5 μg/μl). A pool of 30 RGDV-positive males was used for each replicate in RT-qPCR and western blot assays, respectively. The experiment was conducted in at least three replicates for RT-qPCR and western blot assays. To analyze effect of RGDV infection on PO activity, the reproductive organs dissected from approximate 100 newly emerged males were homogenized with the His-Mg buffer (0.1 M histidine, 0.01 M MgCl₂, pH 6.2) buffer in liquid nitrogen. The supernatant was gently mixed with 1 mM dopamine in 10 mM Tris-HCl buffer (pH 8.0) in a 96-well plate at room temperature for 5 min. Enzyme activity was measured using the phenoloxidase kit (Geruisi, G0146W) according to the manufacturer’s protocol. To analyze the effect of M. luteus infection on PO activity, freeze-dried M. luteus was dissolved in water, and then microinjected in dose of ~23 ng/leafhopper into newly emerged males. At 24-h post microinjection, the reproductive organs of approximate 100 RGDV-infected or M. luteus-treated males were dissected and tested for PO activity.
We then tested the effect of knockdown of PPO or HongrES1 expression on PO activity and RGDV infection. The newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population were microinjected with dsGFP, dsPPO or dsHongrES1 (~200 ng/leafhopper). The male reproductive organs of these tested leafhoppers were individually collected and dissected for RT-qPCR and western blot assays to determine the effect of dsRNAs on the expression levels of HongrES1, PPO, or RGDV P8, and the conversion of PPO to active PO, as well as PO activity. A pool of 30 males was used for each replicate in RT-qPCR and western blot assays, respectively. A pool of 100 males was tested for each replicate in PO activity. The experiment was conducted in three replicates for RT-qPCR and western blot assays, as well as PO activity tests.

A blue LED headlamp (Topme, purchased from www.amazon.com, advertised as a fishing headlamp) or a multi-color LED flashlight (Lumenshooter, purchased from www.amazon.com, advertised as a tactical flashlight) was used for GFP illumination. To reduce stray light in the green and yellow wavelengths, theater stage gel lighting film (Rosco #4990, CalColor Lavender) was cut and inserted between the LED and the focusing lens of the headlamp. Rosco #14 (Medium Straw) and #312 (Canary) theater stage lighting gel film were used individually or in combination as emission filters. The LED lamp was held at approximately a 45-degree angle above and within 3–6 inches of the specimens. Emission filters were placed between the acrylic platform and the clip-on lens. For imaging of red fluorescent proteins, Rosco #88 (Light Green) and #89 (Moss Green) were used for illumination (excitation filters) along with #19 (Fire) as an emission filter. All Rosco filters were purchased from www.stagelightingstore.com or www.bhphotovideo.com. A Vernier Emissions Spectrometer (VSP-EM, www.vernier.com) was used to assess the effects of excitation filters. Vernier Spectral Analysis software (version 4.11.0–1543) was used to acquire and convert data to .csv format, which was then imported into Microsoft Excel and subsequently used to make plots using GraphPad Prism (version 7.0, GraphPad). A Vernier LabPro coupled to a light sensor (Vernier LS-BTA) was used to assess light intensity of blue LED flashlights in combination with Vernier Logger Pro 3 software (version 3.16.2). A multimeter (Amprobe 30XR-A) was used to measure the current draw between the battery and LED on the blue LED headlamp.

A complete parts list, design, and assembly information is available in the Supplementary Methods. Briefly, cut plywood or composite material were assembled with a plexiglass (polycarbonate or acrylic) surface. Holes were drilled into the plexiglass to provide a clear viewing port for the smartphone camera. An additional plexiglass piece was cut to serve as a moveable stage platform immobilized by washers and clamps. A clip-on macro lens (Lieront, purchased from www.amazon.com, advertised as 25X macro) was used to add magnification to smartphone images for fluorescence. A USAF 1951 Test Chart (www.edmundoptics.com, Catalog #3857) was used to determine the maximum image resolution achieved. After the image was acquired with maximal digital zoom, we determined the smallest lines that could be clearly separated and the lp/mm they represent (x). Resolution (in microns) was calculated as follows: µm = 1000 / (x * 2).