Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye.To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 , IL1F7 (skin), ALAS2 (blood), MMP10 , HTN3  and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive, screening of biological evidence. Importantly, messenger RNA markers are surprisingly stable in the dried state in that they can be successfully detected in environmentally impacted and partially degraded samples40.Identification of the tissue source of biological material present on individuals, evidentiary items and at crime scenes can be crucial to a fuller understanding of the circumstances pertaining to a crime. However traditional protein-based body fluid identification methods use a variety of labor intensive and technologically diverse techniques that do not permit the identification of all body fluids and tissues. Thus there remains a need to develop definitive molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues. Although protein35, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the time required for analysis. For qRT-PCR assays, only 3 or 4 markers can be included in a single reaction since each marker requires a different fluorescent dye.Current mRNA body fluid identification assays typically involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms44, methylation analysis46, copy number variant confirmation49, clone characterization50 and as an alternative for gel electrophoresis51. As its name suggests, HRM is a technique that permits the identification of specific PCR products (i.e. amplicons) by their melting temperature (Tm). An amplicon’s precise melting temperature is dependent on its sequence, length and the ionic strength of its environment and can be measured post-PCR to within 0.1°C with specialized software and hardware. HRM assays require only the use of unlabeled PCR primers and a single saturating intercalating fluorescent dye (e.g. Eva Green). The latter provides enhanced sensitivity by ensuring that all available double stranded DNA binding sites are saturated during the melting process. After amplification the amplicon is melted slowly by increasing the temperature and a loss of fluorescence occurs as the DNA strands separate and release the bound saturating dye into solution. By measuring the negative first derivative of fluorescence (F) with respect to temperature (T) (-dF/dT) a distinct and characteristic melt curve is obtained with the peak maximum representing theTm. Importantly, due to the high resolution nature of the amplicon melt analysis, it is possible to perform multiplex analysis of several amplicons in one tube52.In an attempt to simplify mRNA profiling assays, and to reduce the time and cost of analysis, we have investigated alternative technology solutions for mRNA profiling, particularly High Resolution Melt analysis (HRM). HRM is a rapid and relatively cheap bio-analytical method that was initially developed for sequence variant screening  but which has also found utility in single nuclear polymorphism (SNP) typingHere we describe the development and initial performance evaluation of singleplex and multiplex RNA-based HRM assays for the identification of the commonly encountered forensically relevant body fluids and tissues.® , Zenbio , and Zyagen . Cellular skin samples were collected by swabbing human skin or a touched object surface with a sterile water pre-moistened sterile swab. All samples were stored at -20°C or at room temperature until needed. A 50 μl stain or a single cotton swab was used for RNA isolation.Body fluids were collected from volunteers using procedures approved by the University’s Institutional Review Board. Informed written consent was obtained from each donor. Blood samples   were collected by venipuncture into additive-free vacutainers and 50 μl aliquots were placed onto cotton cloth and dried at room temperature. Freshly ejaculated semen  was provided in sealed plastic tubes and stored frozen. After thawing, the semen was absorbed onto sterile cotton swabs and allowed to dry. Buccal samples   were collected from donors using sterile swabs by swabbing the inside of the donor’s mouth. Semen-free vaginal secretions  and menstrual blood  were collected using sterile cotton swabs. Human skin total RNA was obtained from commercial sources: Stratagene/Agilent Technologies , Biochain32. Briefly, 500 μl of pre-heated (56°C for 10 minutes) denaturing solution  was added to a 1.5 mL Safe Lock tube extraction tube  containing the stain or swab. The samples were incubated at 56°C for 30 minutes. The swab or stain pieces were then placed into a DNA IQTM spin basket , re-inserted back into the original extraction tube, and centrifuged at 14,000 rpm  for 5 minutes. After centrifugation, the basket with swab/stain pieces was discarded. To each extract the following was added: 50 μl 2 M sodium acetate and 600 μl acid phenol:chloroform (5:1), pH 4.5 (Ambion by Life Technologies). The samples were placed at 4°C for 30 minutes to separate the layers and then centrifuged for 20 minutes at 14,000 rpm . The RNA-containing top aqueous layer was transferred to a new 1.5 ml microcentrifuge tube, to which 2 μl of GlycoBlue™ glycogen carrier (Ambion by Life Technologies) and 500 μl of isopropanol were added. RNA was precipitated for 1 hour at -20°C. The extracts were then centrifuged at 14,000 rpm . The supernatant was removed and the pellet was washed with 900 μl of 75% ethanol/25% DEPC-treated water. Following a centrifugation for 10 minutes at 14,000 rpm , the supernatant was removed and the pellet dried using vacuum centrifugation (56°C) for 3 minutes. Twenty microliters of pre-heated (60°C for 5 minutes) nuclease free water (Ambion by Life Technologies) was added to each sample followed by an incubation at 60°C for 10 minutes. Samples were used immediately or stored at -20°C until needed. All extracts were DNase treated to remove residual DNA using the Turbo DNA-free™ kit (Applied Biosystems (AB) by Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. RNA extracts were quantitated with Quant-iT™ RiboGreen® RNA Kit  as previously described32. Fluorescence was determined using a Synergy™ 2 Multi-Mode microplate reader .Total RNA was extracted from blood, semen, saliva, vaginal secretions, menstrual blood and skin using a manual organic RNA extraction (guanidine isothiocyanate-phenol-chloroform mixture)All samples were reverse transcribed using the High Capacity cDNA Reverse Transcription kit (AB by Life Technologies) according to manufacturer’s protocols. The desired total RNA input was reverse transcribed in a 20 μl RT reaction volume . If no quantitative value was obtained or quantitation was not performed, an aliquot of the total RNA extract was used (14.2 μl). A reverse transcription negative reaction  was performed for each sample.® HRM™ PCR kit . Each 25 μl reaction included: 1 × HRM master mix , dNTPs and MgCl2), 2.8 μM primers , using the following cycling conditions: 95°C 5 min, followed by 45 cycles of 94°C 10 sec, 55°C 30 sec, 72°C 10 sec. HRM analysis was performed using a 65–90°C temperature range, with +0.1°C increments .Singleplex, Duplex, Triplex Assays: HRM assays were performed using the Type-Itprimers , 2 μl of® HRM™ PCR kit . Each 25 μl reaction included: 1X HRM master mix  of Q-solution, dNTPs and MgCl2), 0.56 – 1.4 μM primers , using the following cycling conditions: 95°C 5 min, followed by 45 cycles of 94°C 10 sec, 57°C 40 sec, 72°C 20 sec. HRM analysis was performed using a 73–90°C temperature range, with +0.1°C increments .‘All Body Fluids’ Hexaplex Assay: HRM assays were performed using the Type-Itprimers  32, MMP10 32, HTN3 32, TGM4 (semen)53, IL19 29 and IL1F7 (skin)28 (Tms. Samples from two donors of each body fluid of interest were used to estimate theTm of each of the selected markers. The observedTms are listed in® HRM™ kit) utilizes a 55°C annealing temperature which may explain why this second product is detected using this platform. Additional work would be needed to conclusively determine if this second peak is in fact MMP3 and to determine if the primer sequences can be modified to be MMP10-specific. MMP10 is also the only marker in the body fluid set in which HRM DNA products were observed, one at 78.5°C and another at 83.1°C (kin)28 . We perf 83.1°C . These wTms were similar or overlapping (Tm(s) for the body fluid of interest was observed, with the exception of the menstrual blood samples  in the lower temperature ranges for the TGM4 and IL19 melt curves in the semen/saliva and vaginal/skin duplex assays, respectively . The art20. Enzymes of the matrix metalloproteinase (MMP) family that play a role in the tissue remodeling that takes place during menstruation are spatiotemporally expressed in endometrial tissue55. Therefore, it is possible that increased levels of MMP10 may be present just prior to menses. The time during the reproductive cycle in which the blood samples in this study were collected was not known. Further work will be needed to evaluate MMP10 expression levels throughout the female reproductive cycle to determine if any trends can be identified amongst numerous donors.A number of different donors  were observed for the body fluids or tissues of interest for the various duplex assays . HoweverEvidentiary items of unknown body fluid/tissue origin would be analyzed using each of the three duplexes in order to determine which body fluids or tissues, or combination thereof, are present. We performed such a process during our evaluation of the body fluid duplexes by taking single source samples of known tissue provenance and testing them against the three duplex HRM assays. Interestingly the duplex HRM assays were able to successfully identify contaminated single source samples. A set of reportedly single source vaginal secretions samples were evaluated using each of the three duplex assays to check that no other cross reacting body fluids would be detected with the assay. As can seen from56.The performance of the duplex assays with a limited number of simulated casework samples was evaluated. The mock casework samples were designed to represent possible casework scenarios including digital penetration and sexual assault . The simulated casework samples included (i) a swab of the surface of male fingers after digital vaginal penetration of a female participant and (ii) a swab of the inside of male underwear worn 3 hours after sexual intercourse in order to attempt to detect possible vaginal secretions that might have been transferred from the penis after a sexual assault. An evaluation of potential transfer of vaginal secretions to male underwear worn after intercourse was selected to represent a sexual assault case rather than vaginal swabs since we had previously demonstrated the ability to detect semen in vaginal swabs (described above). For both samples  both vaginal secretions (IL19) and skin (IL1F7) were detected . These rWe evaluated the ability to detect blood and semen in two person body fluid mixtures as an example of a sample admixture type encountered in casework. We prepared blood-semen mixture samples using a constant amount of blood (50 μl) and decreasing amounts of semen . Each admixed sample was evaluated with the three duplex HRM assays . Blood . The int57, finding biomarkers capable of unequivocally differentiating and identifying each of the three cell types commonly found in casework  is challenging. We have recently had success in the identification of highly specific RNA biomarkers for vaginal secretions and skin29 that, in combination with well-characterized saliva markers such as HTN3 or STATH, are capable of differentiating epithelial cells. Encouraged by the success in developing duplex HRM assays, we conceived and formulated a triplex assay for the identification of epithelial cell containing fluids and tissues . The assay employs the HTN3 , IL19  and skin (IL1F7) RNA biomarkers , TGM4 (semen) and IL1F7 (skin). Nevertheless we sought to develop an ‘all body fluids’ hexaplex system using alternative highly specific biomarkers or primer modifications to intentionally alter theTm value of the RNA amplicon.The availability of a hexaplex assay that would permit a single tube identification of all of the common forensically relevant biological fluids and tissues  is desirable. The development of such an HRM assay is challenging, however, since the number of biomarkers, or sometimes the choice of biomarker, is dictated by the need to assure non-overlappingTm value, sensitivity and specificity28. A CCL27Tm value of 84.8°C no longer presented a conflict with MMP10 (81.9°C). However, this was close to theTm value of ALAS2 (85.8°C). Sequence modification of the reverse primer for ALAS2 permitted a shift of the observed ALAS2Tm value to 86.8°C and therefore resolved any potential overlap with CCL27. The use of an alternative reverse primer sequence for IL19 also resulted in a new observed averageTm value of 78.6°C and therefore resolved the conflict between MMP10 and IL19. The remainingTm conflict was between MMP10 and TGM4. The use of a modified MMP10 forward primer sequence and the inclusion of a “GGGGG” non-template addition to the MMP10 primer set resulted in a sufficient separation of MMP10 and TGM4Tm values . With these modifications a singleTm value for MMP10 was also observed. With the success of these loci and primer modifications, together with alterations to the PCR cycling parameters, we were able to develop a prototype hexaplex HRM system that was suitable for undergoing a series of preliminary developmental validation studies. The hexaplex system is shown inTm overlap as indicated by measurement of the averageTm value andTm value range (+ 3 standard deviations) . The presence of this DNA product can actually be useful in identifying samples that contain significant amounts of contaminating DNA and could therefore be used as an RNA sample quality control check for all sample types, irrespective of body fluid source. Interestingly, similar previously observed DNA products for MMP10 in the duplex assay were not apparent with the hexaplex system.The above described singleplex, duplex and triplex assays involved the use of the following markers: ALAS2 (blood), TGM4 (semen), HTN3 , IL19 , MMP10  and IL1F7 (skin). Our initial efforts were focused on attempting to include all of these biomarkers in the hexaplex system. However attempts at suitably modifying the IL1F7 primers were unsuccessful and resulted in the requirement for an alternative biomarker for skin. We selected CCL27 as a replacement for IL1F7 based on itsations) . A DNA-sations) ,Table 3We carried out initial performance checks on the hexaplex’s specificity. Single source body fluid and tissue samples  were tested. The correct biomarker was identified, except for a small number of false negative results with semen (one sample) and saliva (two samples) . SimilarData Set 1. Tm Values for HRM duplex assay specificity This data set contains the individual Tm values for the specificity testing of the vaginal/skin, blood/menstrual and saliva/semen HRM duplex assay specificity presented in Table 2. Individual donors are listed according to body fluid or tissue type with the Tm values (oC) recorded under each biomarker. IL19 = vaginal biomarker, ALAS2 = blood , HTN3 = saliva biomarker, IL1F7 = skin biomarker, MMP10 = menstrual blood, TGM4 = semen biomarker, n.d. = not detected in body fluid of interest, empty cell = no detectionData Set 2. Tm Values for ‘All Body Fluids’ Hexaplex Assay Specificity This data set contains the individual Tm values for specificity testing of the ‘all body fluids’ hexaplex HRM assay presented in Table 4. Individual donors are listed according to body fluid or tissue type with the observed Tm values recorded under each biomarker. n.d. = not detected in body fluid of interestClick here for additional data file.Tm Values for HRM duplex assay specificity.Data Set 1.Tm values for the specificity testing of the vaginal/skin, blood/menstrual and saliva/semen HRM duplex assay specificity presented inTm values (°C) recorded under each biomarker. IL19 = vaginal biomarker, ALAS2 = blood, HTN3 = saliva biomarker, IL1F7 = skin biomarker, MMP10 = menstrual blood, TGM4 = semen biomarker, n.d. = not detected in body fluid of interest, empty cell = no detection.This data set contains the individualTm Values for ‘All Body Fluids’ Hexaplex Assay Specificity.Data Set 2.Tm values for specificity testing of the ‘all body fluids’ hexaplex HRM assay presented inTm values recorded under each biomarker. n.d. = not detected in body fluid of interest.This data set contains the individualMessenger RNA profiling with a battery of highly specific biomarkers can be used to positively identify all of the commonly found forensically relevant body fluids and tissues. In this work, in order to simplify mRNA profiling assays and to reduce the time and cost of analysis, we have developed a number of prototype multiplex high resolution melt (HRM) assays for the identification of blood, semen, saliva, vaginal secretions, menstrual blood and skin. With respect to critical post-cDNA reagents, the HRM assays require only the use of unlabeled PCR primers and a single intercalating saturating fluorescent dye (Eva Green). In terms of hardware and software a real time instrument with HRM capabilities is required and a number of manufacturers make such instruments. Each body-fluid specific marker can easily be identified by the presence of a distinct melt peak.The ability to multiplex different combinations of RNA biomarkers as evidenced by the compatibility of the markers in duplex, triplex and hexaplex assays indicates that there exists an opportunity for laboratories to customize HRM assays to suit their specific needs. For example, if the intended use of the HRM assays is to rapidly screen sexual assault evidence, it may be desirable to only incorporate biomarkers for the identification of semen and vaginal secretions or, alternatively, semen, vaginal secretions and saliva. Some may want an epithelial marker assay such as the triplex that can identify and differentiate saliva, vaginal secretions and skin. However, some laboratories may want to utilize HRM assays for the identification of all forensically relevant body fluids.Tm. It may also be possible to alter theTm of the existing markers in the assay by changing the primer location. However, any changes to primer sequences would require additional studies to evaluate primer efficiency and to ensure that no changes to sensitivity or specificity are observed.We recognize that the prototype HRM assays may need to be further optimized  using a larger and more varied sample set. For example, the semen/saliva duplex assay should be further optimized in order to reduce or eliminate artifacts such as the broad peaks/humps observed in the HTN3 region arising from the TGM4 melt curve. If these artifacts cannot be eliminated it may be necessary to replace TGM4 with another suitable semen marker with a melt curve profile that does not interfere with the interpretation of HTN3 data or to replace HTN3 with another saliva marker that has a higherThe principal advantages of HRM over current mRNA body fluid identification assays that employ capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms are the not insignificant ones of cost and time. Both CE and qRT-PCR platforms require the use of expensive fluorescently labeled primers or probes whereas HRM uses unlabeled primers. CE-based assays require separate amplification and detection steps thus increasing the cost and time required for analysis. Post RNA extraction and cDNA formation, the closed tube HRM assay takes ~ 2 hours to perform which is similar to qRT-PCR assays. However for qRT-PCR assays, only 2 or 3 markers can be included in a single reaction since each marker and internal control requires a different fluorescent dye whereas HRM can multiplex at least 6 markers.In summary, this proof of principle work describes the design and testing of a number of mRNA HRM assays that, after further validation and optimization, might prove useful in an operational casework setting. The principal advantages of HRM in terms of timeliness and cost may facilitate the technology transfer of mRNA profiling methodology into forensic casework.Body fluids were collected from volunteers using procedures approved by the University’s Institutional Review Board. Informed written consent was obtained from each donor. The authors have satisfactorily addressed all concerns/comments we have initially raised.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Hanson and Ballantyne describe the development of HRM-based single- and multiplex assays for the identification of forensically relevant cell types. These assays are potentially a rapid and relatively inexpensive alternative for mRNA profiling methodologies such as qRT-PCR assays and CE analysis of fluorescently labeled RT-PCR products.The research is scientifically sound and follows a well-designed stepwise approach. The study is focused at assay development and specificity testing. Known markers were chosen  that show good specificity with the body fluids tested.The rationale, results and interpretation of the data are well-described although some figures could be improved (if possible) by using different colors: the use of pink and red to discriminate MMP10 and ALAS2 in Fig2A is somewhat challenging to the eye, and when yellow is used  I found some details quite hard to discern.The authors state ‘the results demonstrate the potential use of HRM assays for the screening of biological evidence’ and ‘we describe the development and initial performance evaluation of the developed HRM assays’. This raises the question what would be needed before HRM assays would be suited for casework?In our laboratory, numerous cases have been analyzed using end-point RT-PCR multiplexes on both cell and organ typing detected by CE. These cases have taught us that most/many forensic casework samples will be 1) mixtures of various cell types and 2) of limited stain size. Thus, besides good specificity, sensitivity and the ability to handle  mixtures are important performance characteristics for RNA assays. The capacity to analyze unequal mixtures will depend on the analysis method. When using CE platforms for instance, high mixture components may result in pull-up signals and artifact peaks that can mask the presence of the minor components or complicate RNA profile interpretation.Thus, I would like to invite the authors to comment on the expected performance of HRM assays when analyzing unequal mixtures.Furthermore, as this initial study did not yet assess sensitivity, I am interested in the authors’ thoughts on the sensitivity that they perceive for HRM-based RNA assays.Overall, I compliment the authors with this study and the novelty they bring to the forensic field.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. "The rationale, results and interpretation of the data are well-described although some figures could be improved (if possible) by using different colors: the use of pink and red to discriminate MMP10 and ALAS2 in Fig2A is somewhat challenging to the eye, and when yellow is used  I found some details quite hard to discern."The colors are used to aid visually distinguishing each body fluid. There are always variations between computers and printers, and therefore the original color selected in the instrument software is not always as clearly represented. We also labeled each biomarker so that its location would be clearly indicated on each diagram. We will keep color selection in mind for future publications. "The authors state ‘the results demonstrate the potential use of HRM assays for the screening of biological evidence’ and ‘we describe the development and initial performance evaluation of the developed HRM assays’. This raises the question what would be needed before HRM assays would be suited for casework?"A full developmental validation in accordance with SWGDAM guidelines would be required. This would include a more comprehensive evaluation of sensitivity, specificity, reproducibility and performance with mixtures and mock casework samples. In our laboratory, numerous cases have been analyzed using end-point RT-PCR multiplexes on both cell and organ typing detected by CE. These cases have taught us that most/many forensic casework samples will be 1) mixtures of various cell types and 2) of limited stain size. Thus, besides good specificity, sensitivity and the ability to handle  mixtures are important performance characteristics for RNA assays. The capacity to analyze unequal mixtures will depend on the analysis method. When using CE platforms for instance, high mixture components may result in pull-up signals and artifact peaks that can mask the presence of the minor components or complicate RNA profile interpretation."Thus, I would like to invite the authors to comment on the expected performance of HRM assays when analyzing unequal mixtures."A blood-semen mixture, with decreasing amounts of semen in each mixture, was evaluated using the duplex assays. Both body fluids were correctly identified in each of these mixtures. Successful detection of both components of a saliva-vaginal admixed sample was also observed for the hexaplex assay. While these were not large-scale comprehensive mixture studies, we are hopeful that the HRM assays will permit the ability to identify all components in admixed body fluid samples. Similar to the experience of the referee with CE based assays, It is likely that some mixture types will be challenging to define because of the particular mixture composition or because one or more of the components are present as a minor fraction. In our experience with CE as well, highly abundant biomarkers tend to dominate detection and can mask the presence of a minor component. It will be interesting to see with future studies whether the use of the HRM assays will permit an improved detection of minor component fluids or tissues. So while more comprehensive mixtures studies are needed , we are hopeful that the successful initial results presage the efficacy of HRM assays to perform reasonably well with mixtures.  [See also the response to the first referee report]. "Furthermore, as this initial study did not yet assess sensitivity, I am interested in the authors’ thoughts on the sensitivity that they perceive for HRM-based RNA assays."We performed initial sensitivity studies for each of the HRM duplex assays. The sensitivity of each of the duplex assays was evaluated by testing a range of input total RNA, from 15pg to 100ng . For the blood/menstrual blood duplex assay, both biomarkers (ALAS2 and MMP10) were detected using picogram quantities of input total RNA ). For the saliva/semen assay, both biomarkers (HTN3 and TGM4) were detected using as little as 25pg input. Using 15pg input, HTN3 was also detected but not TGM4. Therefore the sensitivities of these two duplex assays appear to be highly sensitive, requiring only picogram levels of input total RNA. Additionally, it should be noted that the total RNA input amounts listed represent the amount used in the RT reaction. Detection of product in the real time assays is performed with 1/10th of the RT input amount (2ml cDNA/20ml reaction). For the vaginal secretions/skin duplex assay, 5ng of input RNA (still only 500pg for detection) was required for IL19. Skin was successfully detected with as little as 100pg total RNA RT input. Since only one donor was tested for this initial sensitivity work, the actual sensitivity limits need to be determined with additional testing. For vaginal secretions, since no human specific RNA quantitation method is currently available, there is always a potential for the obtained RNA quantitation value to be artificially higher due to the presence of endogenous bacteria. Therefore, the total RNA input used for these samples may not be fully accurate which will then affect the sensitivity results. But overall, these assays appear to require picogram levels of input total RNA, which would make them suitable for use with forensic, samples. An initial sensitivity check for the hexaplex assay was also performed . The body fluids were detected down to varying lower input limits: blood (ALAS2) - 25 pg; menstrual blood (MMP10) - 250 pg; skin (CCL27) - 1ng; semen - 5ng; vaginal secretions – consistently with 1 ng or more input (variable detection in the 5pg to 1ng range); saliva (HTN3) – not determined at this time.While we performed this preliminary sensitivity evaluation, it was not included in the manuscript since only one donor was evaluated. More comprehensive sensitivity studies, particularly for the hexaplex system, will be included in a future publication describing a comprehensive testing and evaluation of the HRM assay. Hanson & Ballantyne report in this article a novel use for High Resolution Melt analysis of RNA samples in order to identify single or mixed human body fluids (and tissues), namely vaginal secretions, skin, blood, menstrual blood, saliva, or semen using a series of molecular biomarkers: IL19, IL1F7 or CCL27, ALAS2, MMP10, HTN3, and TGM4, respectively. Identification of these body fluids/tissues can be of crucial importance in forensic sciences and criminal cases. The methodology appears to be quite specific and versatile, and according to the authors, less expensive and time-consuming compared to similar mRNA-based platforms such as quantitative real-time polymerase chain reaction or capillary electrophoresis. Remarkably, the technique can allow the identification of up to 6 body fluids in one single reaction .The paper is very well written, scientifically sound and their results are very clear and adequately interpreted and discussed. I support its approval in the journal F1000 Research. I also have several comments to be eventually addressed:Would there be a way to differentiate between blood and menstrual blood samples? Would that be something important to achieve?Have the authors used (or planned to use) this technique with samples such as sweat, urine, or hair? Additionally, could the technique differentiate human from animal samples (using the same biomarkers)?Can the authors give a little bit more information about the size of the amplicons generated by PCR for the different biomarkers employed and also about the design of the primers employed?How reproducible is the assay for one sample coming from an individual?We noticed that when you compare tables 2 and 4, there is a significant improvement of the specificity using the IL19 marker in the hexaplex assay. As mentioned by the authors, they have used a different reverse primer for IL19 in the hexaplex assay. We wonder whether this new reverse primer can also improve the specificity for IL19 to differentiate vaginal secretions from menstrual blood also in single, duplex, or triplex analyses.Regarding the possible amplification of MMP3 by MMP10 primers in the menstrual blood samples under certain experimental conditions. I think it could be important to test that this is indeed the case; maybe by designing MMP3 specific primers that confirm this particular peak or eventually sequencing the products that were generated.Finally, I got the impression from the experiments in which the authors mixed blood with decreasing amounts of semen that the technique does not necessarily reflect quantitative trends. Is that indeed the case? Could quantitation be ultimately important in a forensic case? We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Would there be a way to differentiate between blood and menstrual blood samples? Would that be something important to achieve?The described hexaplex assay has a biomarker for both blood and menstrual blood. Therefore they will be distinguished using this assay. However, menstrual blood will also contain varying amounts of peripheral blood and therefore both the blood (ALAS2) and menstrual blood (MMP10) biomarkers may be present in menstrual blood samples. If the sample is strictly peripheral blood, only the ALAS2 (blood) biomarker would be present and the menstrual blood (MMP10) marker would be absent. This can be important in some cases where intimate contact is implied or suggested based on the circumstances of the case and a suspect argues that it is blood from the nose of the victim, for example, during an assault but not a sexual assault. The identification of menstrual blood as the source would indicate more intimate contact. Additionally during sexual assault investigations, peripheral blood could indicate that trauma had taken place to the female reproductive system, and therefore a determination that the blood present is not simply menstrual blood from normal menstruation could be probative.Have the authors used (or planned to use) this technique with samples such as sweat, urine, or hair? Additionally, could the technique differentiate human from animal samples (using the same biomarkers)?We have not yet used this technique with other types of samples such as sweat, urine or hair. The HRM approach described here could certainly be utilized for the identification of other forensically relevant materials if suitable biomarkers were identified. This could also include internal organs and tissues tissues such as brain, heart, etc. This work was focused on those body fluids and tissues commonly found at the crime scene, but the approach could certainly be used for other fluids and tissues in the future. Can the authors give a little bit more information about the size of the amplicons generated by PCR for the different biomarkers employed and also about the design of the primers employed?All of the primers were designed using Primer 3 software. Primers were designed to either span exon-exon boundaries (no DNA amplification) or designed to be in separate exons (so that the DNA product size would be different than the RNA product size due to the inclusion of an intron). Tables 1 and 3 have been updated to include amplicon size and also the exon location of the primers. How reproducible is the assay for one sample coming from an individual?The assay is highly reproducible within an individual. Typically in a single experiment the Tm curves from the same individual are exactly superimposable upon one another The donors used in this study were also used repeatedly throughout testing, with both separate RNA extractions and multiple RT-PCR’s from the same individual extract being employed. No significant differences in Tm were observed. We noticed that when you compare tables 2 and 4, there is a significant improvement of the specificity using the IL19 marker in the hexaplex assay. As mentioned by the authors, they have used a different reverse primer for IL19 in the hexaplex assay. We wonder whether this new reverse primer can also improve the specificity for IL19 to differentiate vaginal secretions from menstrual blood also in single, duplex, or triplex analyses.Menstrual blood samples will, by nature of the source of the material, contain varying amounts of vaginal secretions. Therefore IL19 in a singleplex assay still will never provide sufficient resolution to distinguish between a vaginal and menstrual sample, as both could contain vaginal secretions. While every menstrual blood sample may not contain sufficient levels of vaginal material to result in IL19 detection, vaginal secretions will still be present in such samples to a varying extent. Therefore a menstrual blood-specific biomarker is typically needed to definitively identify the presence of menstrual blood. A duplex reaction with IL19 and MMP10 for example would permit a determination of vaginal secretions and menstrual blood. Vaginal secretions only samples should have only IL19, whereas menstrual blood samples could contain only MMP10 or a mixture of MMP10 and IL19.Regarding the possible amplification of MMP3 by MMP10 primers in the menstrual blood samples under certain experimental conditions. I think it could be important to test that this is indeed the case; maybe by designing MMP3 specific primers that confirm this particular peak or eventually sequencing the products that were generated.It is certainly of interest to determine whether the additional product is in fact MMP3 and to determine if specific MMP10 and MMP3 primer sets could be distinguished. This will be the subject of future work, although it is important to note that this uncertainty does not interfere with the accurate interpretation of the body fluid results using the current assay.   Finally, I got the impression from the experiments in which the authors mixed blood with decreasing amounts of semen that the technique does not necessarily reflect quantitative trends. Is that indeed the case? Could quantitation be ultimately important in a forensic case?In our evaluation so far of data from the HRM assays, the height of the melt peak, somewhat surprisingly, does not appear to correlate with the amount of analyte.  More in depth studies need to be performed in order to determine possible reasons for this. It should be noted that the melt peak is the first derivative of fluorescence with temperature. It might be the case that other transforms of the data other than the first derivative might prove to be better correlated with analyte input. The ability to make a relative quantitative assessment of the amount of each body fluid present in an unknown sample would be useful. The RNA quantitation method we employ, namely the binding of the fluorescent dye RiboGreen, provides an overall assessment of the amount of RNA present, but cannot provide quantitative information regarding the amount of each fluid present if the sample contains more than one body fluid. While a quantitative assessment is not critical for a determination of the presence or absence of a body fluid, a quantitative assessment could be provide important probative information for admixed body fluid samples.


RNA editing is an important cellular process by which the nucleotides in a mature RNA transcript are altered to cause them to differ from the corresponding DNA sequence. While this process yields essential transcripts in humans and other organisms, it is believed to occur at a relatively small number of loci. The rarity of RNA editing has been challenged by a recent comparison of human RNA and DNA sequence data from 27 individuals, which revealed that over 10,000 human exonic sites appear to exhibit RNA-DNA differences (RDDs). Many of these differences could not have been caused by either of the two previously known human RNA editing mechanisms—ADAR-mediated A→G substitutions or APOBEC1-mediated C→U switches—suggesting that a previously unknown mechanism of RNA editing may be active in humans. Here, we reanalyze these data and demonstrate that genomic sequences exist in these same individuals or in the human genome that match the majority of RDDs. Our results suggest that the majority of these RDD events were observed due to accurate transcription of sequences paralogous to the apparently edited gene but differing at the edited site. In light of our results it seems prudent to conclude that if indeed an unknown mechanism is causing RDD events in humans, such events occur at a much lower frequency than originally proposed. The accurate transcription of genomic DNA into RNA is essential for carrying out cellular processes, as RNA transcripts are either translated into functional proteins or perform functions directly. However, in humans Despite the demonstrated benefits of RNA editing events, RNA editing is currently viewed as a relatively rare phenomenon, with one comprehensive study identifying only several hundred A→G changes in the human transcriptome Li et al. Because Li et al. only searched their RNA-seq reads against the GENCODE sequences, there are actually three different potential sources of spurious RDDs. First, transcribed sequences paralogous to a GENCODE gene present in the reference genome but not included in the GENCODE predictions of protein-coding genes would be incorrectly inferred to be an RDD if nucleotides differed between the two sequences. Second, even ensuring that sequences are unique in the reference genome does not ensure that they are the result of RNA editing: paralogous sequences present in the reference genome that contain a single-nucleotide polymorphism in the sampled individual (such that the exact sequence is not present in the reference genome) would also appear to be RDDs. Finally, nucleotide differences in segregating copy number variants In order to determine the extent to which RDD events could be erroneously called due to transcription of paralogous sequences matching RDD sites, we first asked whether RDD calls made by Li et al. P<2.2×10−16; Fisher's Exact Test using paralogy assignments from ref. P<0.001; see We also found that 1,316 additional RDD sites have at least one paralog in the reference genome not containing the RDD nucleotide. However, such paralogs could contain polymorphisms such that the transcription of these sequences would result in the appearance of RDDs, if the polymorphic allele not present in the reference genome matches the RDD nucleotide. Again, this possibility is supported by the large percentage of such paralogs found in genes (86.5%) or transcribed regions of the genome (86.2%). In total, RDD sites are much more likely to have a paralog than an average human gene  that matches the RDD nucleotide. We therefore asked whether Li et al.s' RDD calls for each individual were matched by genomic reads from the same individual, again, a step not taken in the original paper. Because the list of individuals exhibiting each RDD site was not made available, we attempted to recapitulate Li et al.'s results by mapping their RNA-seq data to a database of transcripts containing the 10,210 RDD sites. We used the short-read mapping program BWA Given the low genomic sequencing coverage of many of the 27 individuals P = 0.013; Fisher's Exact Test) when RDDs matching genomic sequence are removed from the set. When all RDDs matching at least one genomic read from any individual are removed, the percentage of A→G events grows to 24.5%, though this increase is not statistically significant. While this strongly suggests that the RDDs matching genomic sequence are spurious, a substantial number of non-A→G events remain. This observation can be interpreted in two ways: either the remaining RDD sites are likely true RNA editing events, or they are also erroneous observations due to accurate transcription of genomic sequences missed because of the relatively low coverage of the sequences examined in our analysis. The latter interpretation is supported by If RNA editing is largely restricted to A→G substitutions, and if the 5,900 RDD sites matching genomic sequence data are truly spurious, then the remaining 4,310 RDD sites in Li et al.'s set should be enriched for A→G changes. This is indeed the case, as the percentage of all RDD sites that are A→G differences increases from 22.8% to 23.5%  or nucleotide B (the edited nucleotide) and at least 10% of these reads matching nucleotide B. Note that these requirements are similar to those used by Li et al. and ourselves to call RDD sites.  We found 5,666 RDD sites (55.5%) meeting these criteria. To ensure that this approach had adequate specificity, we also checked how often these criteria were met by reads matching one of the other two nucleotides. In other words, when we randomly replaced the B nucleotide of an RDD site with one of the two nucleotides not equal to A or B and repeated our test, we found only ∼910 RDD sites (8.9%) meeting these criteria . Similarly, we verified that sequencing error was not responsible for the sizeable proportion of RDD sites present in a given individual that were also matched by at least one genomic read from that same individual. While on average 30.5% of RDD events found in a given individual were matched by a genomic read from that same individual containing the B nucleotide, only 8.2% of RDD events matched a genomic read having one of the other two nucleotides. It should be noted that some reads not matching either the A or the B nucleotide may be the result of additional polymorphisms or paralogs rather than sequencing error. Thus, we may be overestimating the impact of sequencing error.Figure S1The number of RDD events found in each individual. We called RDD sites we found using RNA-seq data from each of the 27 individuals after performing our own mapping and reapplying the original criteria used by Li et al. (TIF)Click here for additional data file.Table S1Detailed list of the 10,210 RDD sites.(XLS)Click here for additional data file.


Cyprinus carpio) fished by line-fishing from fishpond “Lőrinte halastó” located in Veszprém County, Hungary. The complete nucleotide (nt) sequence of the genomic RNA is 9565 nt in length and contains two long - non-in-frame - open reading frames (ORFs), which are separated by an intergenic region. The ORF1 (replicase) is preceded by an untranslated sequence of 827 nt, while an untranslated region of 139 nt follows the ORF2 (capsid proteins). The deduced amino acid (aa) sequences of the ORFs showed only low (less than 32%) and partial similarity to the non-structural  and structural proteins (VP2/VP4/VP3) of virus families in Picornavirales especially to members of the viruses with dicistronic genome. Halastavi árva RNA virus is present in intestinal contents of omnivorous freshwater carps but the origin and the host species of this virus remains unknown. The unique viral sequence and the actual position indicate that Halastavi árva RNA virus seems to be the first member of a new di-cistronic ssRNA virus. Further studies are required to investigate the specific host species (and spectrum), ecology and role of Halastavi árva RNA virus in the nature.A novel positive-sense, single-stranded RNA (+ssRNA) virus  with di-cistronic genome organization was serendipitously identified in intestinal contents of freshwater carps ( Picornavirales contains several important viruses grouped into 5 families: Dicistroviridae, Iflaviridae, Marnaviridae, Picornaviridae and SecoviridaePicorna-like viruses are a loosely defined group of non-enveloped, positive-sense single-stranded RNA (+ssRNA) viruses that are major pathogens of humans, animals, insects and plants x-Pro-Pol module of conserved replication domains) and the other – the IGR (intergenic region) - IRES - for translation of the capsid proteins (VP2-VP4-VP3 and VP1) Based upon the genome organization non-enveloped, positive-sense single-stranded RNA (+ssRNA) viruses have two groups: viruses with mono- or di-cistronic genome characteristics. Iflaviruses, marnaviruses, picornaviruses and secoviruses have monocistronic genome structure with a large open reading frame (ORF) coding a single polyprotein. However, dicistroviruses - the name (dicistro) is derived from the characteristic di-cistronic arrangements of the genome - have two non-overlapping large open reading frames (ORF1 and ORF2). In dicistroviruses, the structural proteins are located at the 3′ end (ORF2) rather than the 5′ end as found in iflaviruses, marnaviruses, picornaviruses and secoviruses. The most outstanding characteristics of dicistroviruses is that they have two internal ribosome entry site (IRES) elements, one for translation of the ORF1 replicase (including 2C-like helicase (Hel), VPg, 3C-like protease (Pro) and 3D-like RNA dependent RNA-polymerase (Pol) in order Hel-(VPg)Rhizosolenia setigera RNA virus (RsetRNAV), Chaetoceros socialis f. radians RNA virus (CsfrRNAV) and Chaetoceros tenuissimus RNA virus (CtenRNAV) and fungoid protist virus (Aurantiochytrium single-straded RNA virus (AsRNAV): previously designated as Schizochytrium single-stranded RNA virus (SssRNAV), respectively Heterosigma akashiwo RNA virus (HaRNAV), a prototype member of Marnaviridae – is infecting unicellular bloom-forming raphidophyte alga Members of dicistroviruses have originally been identified in aphids, leafhoppers, flies, bees, ants, silkworms and shrimps Cyprinus carpio) in Hungary.The first report of metagenomic analyis of RNA viruses in fresh water lake was published in 2009 E value = 4×10−14, identities = 64/179; 36%) using BLASTp searches of the NCBI database. The novel genome sequence was provisionally named as Halastavi árva (in Hungarian = “fishpond orphan” in English) RNA virus .Using non-human entero-R/F screening primers  an approNo significant sequence match was found in GenBank based upon the complete nucleotide sequence. The Halastavi árva RNA virus genome is 9565 nt in length , excludix-Pro-Pol module of conserved replication domains. Except the approximately 350 aa 5′-end, which has no sequence match in GenBank, the ORF1 has characteristic aa sequences of 2C-like helicase/NTP-binding protein , 3C-like cystein protease  and 3D-like RNA-dependent RNA polymerase . The “picorna-like” 2A motif NPGP was not found. Potential tandem repeat sequence Q/HCAIGLFVKDQ/SCLMNKHSLDQ/L was detected at aa position 1116 and 1127 (between 2C-like cystein protease and 3C-like RNA polymerase) corresponding to potential two short (11 aa each) VPg (3B) sequences  and the shrimp virus Taura syndrome virus . Detailed amino acid similarity analysis of ORF1 regions are shown in ORF1 possesses an AUG codon at nt 828 and extends to nt 6278 . ORF1 enequences . These sAurantiochytrium single-stranded RNA virus, YP_398835) dicistrovirus ORF2. Recognizable putative picornavirus-like capsid protein domains were identified by the conserved domain search of the NCBI database  on the Halastavi árva RNA virus with the capsid proteins  of viruses in family Dicistro- and Picornaviridae. In addition, an assumed spherical virus-type peptidase  with high E value (0.13) was also identified at the N-terminal end (from 75–250 aa in VP2) of the putative ORF2 capsid-polyprotein  and Drosophila C virus . The amino acid sequence identity of Halastavi árva RNA virus ORF2 to marine RNA virus JP-A is 28% . Detailed amino acid similarity analysis of ORF2 regions  are shown in ORF2 is in a different frame to ORF1 . It possdatabase . Multiplyprotein . There wCleavage sites of the ORF2 encoded polypeptide were not experimentally determined but estimated by alignment of deduced amino acid sequences. The putative protease-mediated cleavage sites of ORF2 polyprotein are RAFGF/SSPPD (highly conserved motif among dicistroviruses at aa position 339/340) and ESMQ/DPY (at aa position 665/666) between capsid protein VP4/VP3 and VP3/VP1 in Halastavi árva RNA virus, respectively. The predicted amino acid length of VP3 and VP1 are 326 aa and 344 aa. VP2+VP4 are 339 aa long. The cleavage site between VP2 and VP4 is probably between aa260 or aa280, maybe at position aa262/aa263 (Q/T). Based upon this calculation the predicted VP2 and VP4 minor capsid proteins are 262 and 77 amino acid-long, respectively.ORFs 1 and 2 represent 88.7% of the RNA genome. The other 11.3% consists of non-coding regions (UTRs and IGR). The 5′UTR is 827 nts in length and it predicted an extensive secondary structure with several long base-paired hairpins (data not shown). Between ORF1 replicase and ORF2 capsid coding regions Halastavi árva RNA virus has a predicted IGR of 118 nts. The exact length of the functional IGR and the secondary structure of the IGR-IRES remained unknown. Conserved dicistrovirus IGR-IRES-like structural elements in which they were previously reviewed by Nakashima & Uchiumi (2009) were not detected. The length of the 3′UTR in dicistroviruses varies from about 150 to 500 nucleotides. Halastavi árva RNA virus 3′UTR is short (139 nt excluding the poly(A) tail) and has an extensive predicted secondary structure consisted of “T”-like double-stranded hairpins (figure not shown). Similar non-coding sequences had not been found in GenBank.Picornavirales order. Halastavi árva RNA virus showed no close relationship to any existing viruses, an observation consistent with the lack of close resemblance of its genome architecture to that of other known RNA viruses.The base composition of Halastavi árva RNA virus is 26.6% A, 27.4% C, 20.3% G, and 25.7%U; this results in a G+C of 47.7%. In common with other RNA viruses (and host genomes), Halastavi árva RNA virus shows a variety of under- and over-representations of dinucleotide frequencies compared to those expected from its mononucleotide base composition . For exaNucleotide composition analysis (NCA) was used to predict the possible host for Halastavi árva RNA virus. In contrast to previous use of this method Using specific primers designed for Halastavi árva RNA virus 2C-like helicase/3A? region (between nt positions 3265 and 3714) a specific 450 nt long PCR-product was detected for Halastavi árva RNA virus by RT-PCR and sequencing in the intestinal content of a second carp, too, fished from the same fishpond at the same time. The two nucleotide sequences were identical.Cyprinus carpio) in Hungary. Based upon the results of the sequence- and phylogenetic analysis this virus belongs to the order Picornavirales. Halastavi árva RNA virus has no nucleotide sequence match in GenBank based upon the complete nucleotide sequence. Halastavi árva RNA virus has only low and partial amino acid sequence identity to known virus families in Picornavirales. In addition, the putative nonstuctural protein domains and capsid proteins show a mosaic pattern of relationships with sequences from viruses in this order especially in family Dicistroviridae. However, Halastavi árva RNA virus is different from the presently known virus families according to the possible host organism; the nucleotide/amino acid sequence identity; the possible unique structures of UTR/IGR and the AU/GC ratios compared to other viruses in Picornavirales. These data suggests that Halastavi árva RNA virus seems to be the first member of a possible new – previously unknown – family of ssRNA viruses.A novel – possibly non-enveloped - positive-sense single-stranded RNA (+ssRNA) virus  with di-cistronic genome organization was identified and characterized originated from the intestinal content of freshwater carp (Cyprinus carpio) are omnivorous, host assignment of Halastavi árva RNA virus recovered from intestinal contents of these fishes is particularly problematic. As a specific example, we have previously shown that viruses recovered from faeces of a child from Afghanistan which showed an unexpected genome organizations similar to insect dicistroviruses, could be predicted to have an insect host by NCA, in this case also potentially deriving from a dietary source Picornavirales. Further specific studies are required to investigate the specific host species (and spectrum), ecology and role of Halastavi árva RNA virus in the nature.Inferring the host of genetically distinct viruses is problematic especially if they are found in feces. Feces are known to contain viruses that infect host cells and/or bacteriophages, as well as viruses of dietary origins from consumed plants, insects, and animals Dicistroviridae. Several of these viruses contain internal ribosome entry site (IRES) that position the ribosome on the genome, actuating translation initiation even in the absence of known canonical initiation factors The AU ratios of the three diatom viruses with ssRNA genome range 60.4% to 63.7%. In insect dicistroviruses AU ratios range 60 to 64%, while that of HaRNAV and SssRNAV are much lower; 53.1% and 50.2%, respectively. Interestingly, the base composition of Halastavi árva RNA virus is more similar (52.3%) to this last group. On the other hand, Halastavi árva RNA virus genome structure appears to have a polycistronic genome organization similar to that found in viruses in family E value = 10−5). The verification of the presence of this putative peptidase at the N-terminal capsid polyprotein in viruses with di-cistronic genome organization warrants future investigations.During the conserved domain search, a putative spherical virus-type peptidase was identified at the N-terminal end of the ORF2 polypeptide. This type of peptidase is responsible for the cleavage of the viral polyprotein into individual proteins in Rice tungro spherical virus Unexpectedly high number of known and novel viruses particularly +ssRNA viruses were identified in marine and recently in fresh water samples by metagenomic methods Cyprinus carpio) during fish processing. No further samples are available from fishes. Carps were fished by line-fishing from fishpond “Lőrinte halastó” located in Veszprém County, Hungary, in Apr 18, 2010. The specimen was stored at −20°C until RNA isolation.Intestinal contents were freshly collected from the intestine of two freshwater carps . The PCR reaction was conducted under the following conditions: 1 cycle at 94°C for 1 min, 40 cycles of 94°C for 30 sec, 57°C for 30 s, 72°C for 1 min, followed by a final elongation step of 72°C for 5 min.RNA was extracted from 150 µl intestinal content suspension  using TRIzol reagent according to the manufacturer's instructions . Generic Non-HumanEntero-5′UTR PCR primers  were useThe 3′ RACE reaction in brief, cDNA was synthesized using an Oligo dT-anchor primer  for the four reactions from total RNA. The RNA template was degraded with RNaseH, and the cDNA was purified. The 3′end of the cDNA was polyadenilated using terminal deoxynucleotidyl transferase and dATP. The polyA tailed cDNA then amplified in 50 µl final volume using Pfu DNA polymerase, Oligo dT-anchor primer and primers as1b, as2b, as3b and as4b, respectively. The PCR of the 3′ and 5′ RACE experiments was conducted using the following temperature conditions: 1 cycle at 94°C for 30 sec, 35 cycles of 94°C for 35 sec, 50°C for 1 min, 72°C for 5 min, followed by a final elongation step of 72°C for 10 min. The amplicons were subjected to second PCR reaction using upstream antisense primers , the PCR anchor primer and the same PCR thermal program used in the first PCR round . r primer  from totr primer . The 5′RCR round . The ampSamples with any visible amplicons were sequenced directly with the BigDye Terminator Cycle Sequencing Ready Reaction Kit  using the PCR primers by primer walking methods and run on an automated sequencer . Amino acid sequences - reference strains were collected from GenBank database - were aligned by ClustalX (version 1.81) and similarity analysis were performed using GeneDoc 2.7 software A set of 352 virus complete genome or segment sequences longer than 3000 bases selected to be representative of different species, genera and families of positive-stranded RNA viruses classified in the picorna-like viruses were used for NCA NCA used the discriminant analysis program in the statistical package, SYSTAT with default parameters. Sequences were assigned to four host categories, mammal (n = 117), insect (n = 63), plant (n = 167) and fish (n = 5) and frequencies of each mononucleotide and dinucleotide used as predictive factors to infer host ranges of unknown virus sequences from the current study.Table S1Host assignment of control and Halastavi árva RNA virus  sequence by NCA.(DOC)Click here for additional data file.


An important challenge in biology has been to understand how cell-type-specific expression programs are orchestrated through regulated access to chromatin. Knowledge of the interaction between noncoding RNAs (ncRNAs) and chromatin regulators has the potential to help answer such questions, but how ncRNAs target chromatin regulators to specific sites in the genome is not well understood. Recently, Jeon and Lee proposed that DNA-binding proteins act as a bridge between ncRNAs and their target sites in chromatin. In this minireview, we examine their findings and place them in the wider context of how chromatin regulator-RNA complexes are targeted to specific sites in chromatin. A number of noncoding RNAs (ncRNAs) have been demonstrated to play a role in transcriptional or chromatin regulation through their interaction with chromatin-modifying enzymes and transcription factors. Some of these RNAs can be visualised to be associated with specific loci, most strikingly the coating of the inactive X chromosome (Xi) by X inactive-specific transcript (Xist) RNA. Modulation of a number of ncRNAs leads to changes in the targeting of regulatory complexes to specific genomic sites. Mechanisms must therefore be in operation that direct ncRNA-protein complexes to specific sites in the genome.Xic) of the future Xi and progressively coats the chromosome. This is accompanied by the appearance of repressive chromatin modifications, including those catalysed by polycomb repressive complex 2 (PRC2). Xist RNA has been proposed to play a direct role in the recruitment of PRC2 through protein-RNA interaction [in situ hybridization and RT-PCR to distinguish between endogenous and transgenic Xist expression [Xic and Xist RNA, Jeon and Lee found that Xist and YY1 interact in cells and that YY1 interacts with the Xist repeat C region in vitro. Consistent with this finding, an earlier study conducted at the Lee laboratory showed that targeting the C region with locked nucleic acid (LNA) oligonucleotides caused displacement of Xist from the Xi [Xic by simultaneously binding to Xist DNA and RNA [Dosage compensation in female mammals is achieved by silencing one copy of the two X chromosomes, termed 'X-chromosome inactivation' (XCI). The long ncRNA Xist is the key factor in initiating this process . Xist ispression . Transgem the Xi . On the  and RNA   methylation at HOXD and several other sites in the genome in trans [The mechanism identified by Jeon and Lee  may be rin trans . HOTAIR in trans . Authorsin trans  and 'capin trans , respectin trans . The autHOXA locus and interacts with mixed-lineage leukaemia (MLL) H3K4 methyltransferase complexes through WD repeat-containing protein 5 (WDR5) [HOTTIP into contact with downstream parts of HOXA, and it has been proposed that this allows HOTTIP RNA to target WDR5-MLL to downstream genes [trans, including those on different chromosomes.Another potential mechanism involved in the targeting of ncRNAs to distant DNA target sites is the proximity between loci induced by chromosomal looping Figure . The lon5 (WDR5) . Chromosam genes . Chromosam genes -14. Knocam genes . One cou0, and in vitro gel shift and protection assays suggest that it forms a triplex structure with the T0 DNA sequence. In addition to allowing the RNA to associate with rRNA promoters, this triplex structure is recognised by DNMT3B and is required for DNA methylation [Direct interactions between RNA and DNA sequences also provide a potential mechanism by which ncRNAs target chromatin regulators to specific sites Figure . Short phylation . Whethercis as it is being transcribed (Figure cis, and the RNAs directly interact with PRC2 in vitro and in cells [RNA can target transcriptional regulator proteins in d Figure . The 60-in cells .Growing evidence suggests that ncRNAs play an important role in chromatin and transcriptional regulation. How these ncRNAs are localised to specific sites in chromatin is not yet clear, but common themes may be emerging. Jeon and Lee's study  outlinesRT-PCR: reverse transcriptase polymerase chain reaction.The authors declare that they have no competing interests.AK and RGJ wrote this review together, and both read and approved the final manuscript.


Caenorhabditis elegans.An essential stage of neural development involves the assembly of neural circuits via formation of inter-neuronal connections. Early steps in neural circuit formation, including cell migration, axon guidance, and the localization of synaptic components, are well described. However, upon reaching their target region, most neurites still contact many potential partners. In order to assemble functional circuits, it is critical that within this group of cells, neurons identify and form connections only with their appropriate partners, a process we call synaptic partner recognition (SPR). To understand how SPR is mediated, we previously developed a genetically encoded fluorescent trans-synaptic marker called NLG-1 GRASP, which labels synaptic contacts between individual neurons of interest in dense cellular environments in the genetic model organism unc-6 and unc-40 animals despite normal axon guidance and subcellular localization of synaptic components. Additionally, behavioral defects indicate a complete disruption of PHB circuit function in unc-40 mutants. Our data indicate that UNC-40 and UNC-6 function in PHB and AVA, respectively, to specify SPR. Strikingly, overexpression of UNC-6 in postsynaptic neurons is sufficient to promote increased PHB-AVA synaptogenesis and to potentiate the behavioral response beyond wild-type levels. Furthermore, an artificially membrane-tethered UNC-6 expressed in the postsynaptic neurons promotes SPR, consistent with a short-range signal between adjacent synaptic partners.Here, we describe the first use of NLG-1 GRASP technology, to identify SPR genes that function in this critical process. The NLG-1 GRASP system allows us to assess synaptogenesis between PHB sensory neurons and AVA interneurons instantly in live animals, making genetic analysis feasible. Additionally, we employ a behavioral assay to specifically test PHB sensory circuit function. Utilizing this approach, we reveal a new role for the secreted UNC-6/Netrin ligand and its transmembrane receptor UNC-40/Deleted in colorectal cancer (DCC) in SPR. Synapses between PHB and AVA are severely reduced in These results indicate that the conserved UNC-6/Netrin-UNC-40/DCC ligand-receptor pair has a previously unknown function, acting in a juxtacrine manner to specify recognition of individual postsynaptic neurons. Furthermore, they illustrate the potential of this new approach, combining NLG-1 GRASP and behavioral analysis, in gene discovery and characterization. Caenorhabditis elegans, the only organism for which there is a complete synaptic map generated through decades of electron micrograph reconstruction studies, on average only one out of six contacting neurons form synapses .-22.20-22pnlp-1 . We. We44]. P = 0.9; Additional file unc-4 and unc-37 mutants (as previously observed with electron microscopy) [unc-40 and unc-6 were in fact more severe.To determine if the NLG-1 GRASP marker affects synaptogenesis between PHB and AVA, the response of transgene-carrying animals was assayed. If synaptogenesis between PHB and AVA is increased by the NLG-1 GRASP marker, then SDS avoidance should be enhanced in animals with the NLG-1 GRASP transgene compared with non-marker carrying animals. However, wild-type animals expressing NLG-1 GRASP are indistinguishable from wild-type animals without the marker in their response to SDS (roscopy) . Similarroscopy) . If NLG-nlg-1 mutant animals respond similarly to wild-type animals, indicating that nlg-1 is not required for PHB-AVA SPR , some PHB-AVA synaptogenesis persists in the mutant animals. As unc-6(ev400) and unc-40(e271) are likely null alleles, this indicates that other molecules may function in parallel to the unc-6-unc-40 signaling cassette. However, PHB circuit-specific behavior is completely disrupted. The NLG-1 GRASP approach tests the ability to recognize and form synaptic structures with the correct partner, while the circuit-specific behavioral assay tests whether the synapses formed are sufficient for a behavioral response. Thus, the response to SDS may be completely defective because the reduced number of PHB-AVA synapses in unc-40 mutants are insufficient for circuit function.Interestingly, although defects in PHB-AVA synaptogenesis are severe in unc-40 and unc-6 mutants. Persisting contact along approximately 73% of the region of neurite overlap may result from molecules that act in parallel to unc-6 and unc-40 or to other interactions within the nerve bundle. The severe defects in PHB-AVA synaptogenesis are not likely a direct consequence of defects in PHB-AVA neurite contact because unc-7/Innexin mutants have PHB-AVA neurite contact defects as severe as unc-6 and unc-40 mutants, yet display no reduction in synaptogenesis. SPR defects are also unlikely to result from general nerve bundle defasciculation, as fasciculation mutants [On the other hand, adhesion between synaptic partners is only partially disrupted in  mutants ,33 displin vivo localization data indicating that UNC-6 is present in puncta dispersed in the AVA neurite [Cell-specific rescue experiments indicate that UNC-6 acts in postsynaptic AVA neurons, and UNC-40 acts in presynaptic PHB neurons to mediate SPR. Furthermore, overexpression of UNC-6 in AVA neurons is sufficient to promote increased PHB to AVA synaptogenesis. Overexpression of UNC-40 in PHB does not promote additional synapses, consistent with a model in which UNC-6 is a limiting signal for regulating synaptogenesis, and unbound UNC-40 cannot instruct SPR. This model is supported by  neurite , while Uin vivo studies reporting function of these molecules in pre- and post-synaptic partners to promote SPR. In fact, during axon guidance, UNC-6/Netrin is frequently secreted from guidepost cells located in or near intermediate or final target regions, but not from specific postsynaptic neurons [in vitro experiments indicate that vertebrate GPI-linked Netrins (Netrin Gs) can promote synaptogenesis between partners . Howeveiewed in .Our results suggest that UNC-6 functions in close proximity to the postsynaptic membrane, because an artificially membrane-tethered UNC-6 expressed in postsynaptic AVAs can promote synapse formation with PHB neurons. The effects are less robust than with secreted UNC-6, which may be due to decreased activity caused by fusion to a heterologous membrane tether. Support for a short-range adhesive capacity for UNC-6/Netrin comes from studies in the developing mammary gland that indicate that Netrin mediates adhesion between preluminal cells expressing Netrin-1 and cap cells expressing the DCC paralog Neogenin . FurtherWe propose that UNC-6 and UNC-40 precisely specify the region of contact between PHB and AVA for synaptogenesis by recruiting presynaptic vesicles in the distal axon to the PHB neurite membrane that abuts AVA  to describe the population. Similarly, analysis of transgenic rescue and overexpression experiments requires analysis of a statistically significant population of animals . This is prohibitively time-consuming and labor-intensive with current methods such as electron microscopy reconstruction, which requires months to years for each animal. Importantly, the addition of a circuit-specific behavioral assay allowed us to confirm the function of these synapses in live animals with similarly large sample sizes. Therefore, this new approach allows quantitative genetic analysis of SPR in live animals and shows great promise for discovery and characterization of genes that mediate this critical process in neuronal development.NLG-1 GRASP presents unique advantages that made our discovery possible. In this study, NLG-1 GRASP was employed to identify genes that mediate the ability of PHB sensory neurons to recognize and form synapses with AVA interneurons within a dense synaptic region. It would have been impossible to uncover a role for C. elegans variety Bristol, strain N2. Except for strains containing the wyEx2309, wyEx1098, wyEx2871, iyEx82, iyEx83, iyEx84, and iyEx85 transgenes, all strains contain the integrated pnlp-1::mCherry (10 ng/μl), pflp-18::mCherry (5 ng/μl), pgpa-6::nlg-1::spGFP1-10 (60 ng/μl), pflp-18::nlg-1::spGFP11 (30 ng/μl), and podr-1::RFP (20 ng/μl) transgene wyIs157 IV. Strains were maintained by standard methods [Escherichia coli-seeded NGM plates at 20°C.Wild-type strains were  methods . All worage-1(hx546) II, cfi-1(ky651) I, clec-38(tm2035) V, ina-1(gm39) III, mig-10(ct41) III, nlg-1(ok259) X, sdn-1(ok449) X, unc-5(e53) IV, unc-6(ev400) X, unc-7(e5) X, unc-34(gm104) V, unc-40(e271) I, unc-42(e419) V, unc-104(e1265) II, unc-115(ky275) X, unc-129(ev554) IV, and wyIs157 IV. Transgenes maintained as extrachromosomal arrays include two lines used for cell-specific rescue of unc-6(ev400) X: iyEx1 and iyEx5 (prig-3::unc-6 (40 ng/μl), punc-122::RFP (20 ng/μl)), two lines used for cell-specific rescue of unc-6(ev400) X with a membrane-tethered unc-6: iyEx19 and iyEx21 , punc-122:: RFP (20 ng/μl)), two lines used for cell-specific rescue of unc-40(e271) I: iyEx33 and iyEx58 (pnlp-1::unc-40 (10 ng/μl), punc-122:RFP (20 ng/μl)). Extrachromosomal transgenic arrays used for overexpression experiments include two lines used for overexpression of unc-6: iyEx47 and iyEx48 (prig-3::unc-6 (40 ng/μl), punc-122:RFP (20 ng/μl)), two lines used for overexpression of membrane-tethered unc-6: iyEx56 and iyEx60 , punc-122:RFP (20 ng/μl)), and three lines used for overexpression of unc-40: iyEx53, iyEx52 and iyEx54 (pnlp-1::unc-40 (5 ng/μl), punc-122:RFP (20 ng/μl)). The extrachromosomal transgenic array used for presynaptic or postsynaptic localization in wild type, unc-6/Netrin, and unc-40/DCC in Figure wyEx2309 (pnlp-1::mCherry::rab-3 (1 ng/μl), pflp-18::nlg-1::YFP (30 ng/μl), punc-122::RFP (30 ng/μl)), in wild type and cfi-1; unc-42 in Figure wyEx1098 (pnlp-1::mCherry::rab-3 (10 ng/μl), podr-1::RFP (50 ng/μl)), in wild type and unc-6/Netrin in Figure S5A-D,S in Additional file iyEx84 (pgpa-6::syd-2::YFP (30 ng/μl), punc-122::RFP (20 ng/μl)), in wild type and unc-40/DCC in Figure S5E,F,S in Additional file iyEx85 (pgpa-6::syd-2::YFP (30 ng/μl), punc-122::RFP (20 ng/μl)), in wild type, unc-6, and unc-40 in Figure S5G-L,S in Additional file iyEx83 (pgpa-6::GFP::elks-1 (30 ng/μl), punc-122::RFP (20 ng/μl)), and in wild type, unc-6, and unc-40 in Figure S5M-R,S in Additional file iyEx82 (pflp-18::nlg-1::YFP (30 ng/μl), pnlp-1::mCherry::rab-3 (0.5 ng/μl), punc-122::RFP (20 ng/μl)). The extrachromosomal transgenic array used for unc-40 localization in both wild-type and unc-6 mutant backgrounds is wyEx2871 (pgpa-6::unc-40::GFP (35 ng/μl), punc-122::RFP (20 ng/μl)). Extrachromosomal arrays used for rescue or overexpression were tested in the F4 to F10 generations.Mutations and integrated transgenes used in this study include age-1hx46 II, cfage-1hx46 II, cfage-1hx46 II, cfage-1hx46 II, cfpflp-18::mCherry [pflp-18::nlg-1::spGFP11 [punc-122::RFP [podr-1::RFP [pnlp-1::mCherry construct, the nlp-1 promoter was amplified from N2 genomic DNA, adding 5' NotI and 3' XbaI sites, and subcloned into the NotI-XbaI fragment from pttx-3::mCherry [ttx-3 promoter. To generate the pgpa-6::nlg-1::spGFP1-10 construct, the gpa-6 [nlg-1::spGFP1-10 [prig-3::unc-6 construct, the SphI-AscI rig-3 promoter fragment from prig-3::CD4-2::spGFP1-10 [pegl-20::unc-6 [egl-20 promoter. To generate the prig-3::MTunc-6::mCherry  construct, the SphI-AscI rig-3 promoter fragment was subcloned into the SphI-AscI fragment from punc-6::MTunc-6::mCherry , replacing the unc-6 promoter. To generate the pnlp-1::unc-40 construct, site-directed mutagenesis  was used to mutate an internal SphI site in the nlp-1 promoter, and the SmaI-SphI nlp-1 promoter fragment was subcloned into the SmaI-SphI fragment from pmig-13::unc-40 , replacing the mig-13 promoter. To generate the pgpa-6::unc-40::GFP construct, the SphI-SmaI gpa-6 promoter fragment was subcloned into the SphI-SmaI fragment from pmig-13::unc-40::GFP , replacing the mig-13 promoter. To generate the pnlp-1::mCherry::rab-3 construct, the NotI-XbaI nlp-1 promoter fragment was subcloned into the NotI-XbaI fragment from pttx-3::mCherry::rab-3 [ttx-3 promoter. To generate the pflp-18::nlg-1::YFP construct, the SphI-AscI flp-18 promoter fragment was subcloned into the SphI-AscI fragment from popt-3::nlg-1::YFP [opt-3 promoter.The following plasmids and transgenic strains were generated using standard techniques: :mCherry , pflp-18:spGFP11 , punc-12122::RFP , podr-1:r-1::RFP  were prehe gpa-6  promoterpGFP1-10 . To genepGFP1-10  was subc0::unc-6 , replacig-1::YFP , replacipgpa-6::syd-2::YFP construct, pgpa-6::GFP was first generated by amplifying the gpa-6 promoter from N2 genomic DNA, adding 5' NotI and 3' BamHI sites, and subcloning it into the NotI-BamHI fragment of pSM::GFP. The NheI-ApaI fragment from punc-86::syd-2::YFP [pgpa-6::GFP, replacing the GFP cDNA. To generate the pgpa-6::GFP::elks-1 construct, the SphI-SmaI gpa-6 promoter fragment was subcloned into the SphI-SmaI fragment from punc-86::GFP::elks-1 [unc-86 promoter. To generate the pgpa-6::mCherry::rab-3 construct, the SphI-SmaI gpa-6 promoter fragment was subcloned into the SphI-SmaI fragment from pflp-18::mCherry::rab-3 [flp-18 promoter. The pflp-18::nlg-1::YFP construct was generated using a carboxy-terminal YFP Gateway Destination vector [flp-18 promoter. cDNAs in attL containing pDONR201 vector  encoding NLG-1 was recombined into the carboxy-terminal YFP pSM Gateway Destination vector with LR clonase .To generate the d-2::YFP  was then::elks-1 , replaciy::rab-3 , replacin vector  with theC. elegans using a Zeiss Axio Imager.A1 compound fluorescent microscope under 630 × magnification. For phenotypic quantification, all micrographs were taken of larval stage animals: L1 to L2 stage animals  of each NLG-1 GRASP punctum or cluster of puncta, and summing these lengths. Percent synaptic length was calculated by subtracting the sum of the synaptic lengths from the length of overlap, and dividing by the length of overlap in each animal. The percent animals with normal axon guidance was determined by adding the total number of animals that have both PHB axons extending into the ventral nerve cord  and dividing it by the total number of animals assayed. The percentage of gaps in each region was quantified by tallying the number of gaps between parallel neurites in the anterior, medial, and posterior regions of the preanal ganglion and dividing by the total number of gaps.NIH ImageJ  was usedA nose touch behavior assay was developed to test PHB-AVA function by adapting a published dry drop test . An adulWe tested the response of at least 40 adults to the control M13 buffer and at least 40 adults to 0.1% SDS (in M13) to find the individual backing times for both wild-type and mutant or transgenic animals. In strains with PHB or AVA axon guidance defects, animals with wild-type axon guidance were isolated using the Axio Imager. A1 compound fluorescent microscope at the L4 stage; anesthetics cause impaired movement and so no anesthetic was used. The behavior assay was performed on these animals at the gravid adult stage the following day. The relative response index was calculated by dividing the average backing time into SDS by the average backing time into buffer to account for slow movement in mutants or transgenic lines. This calculated value is divided by the same value for wild-type animals to normalize the wild-type response index to 100%.P > 0.05), it is not necessary to follow up with pair-wise comparisons. If the result of the Kruskal-Wallis test is found to be significant, which means that the medians of at least two of the groups differ significantly, it is followed up by a Mann-Whitney u-test, which compares the medians of the two independent groups. If more than one u-test is conducted, the resulting P-values are adjusted for multiple comparisons by the Hochberg method. The Hochberg procedure is a standard procedure applied to adjust for the tendency to incorrectly reject a null hypothesis when multiple comparisons are made, and can only conservatively increase P-values. A multiple comparison procedure is required whenever several conclusions are drawn from the same group of data in order to assure that the overall error rate for a type I error is still bound by the chosen significance level .Median values for relative intensity were compared first by a Kruskal-Wallis test, a nonparametric alternative to ANOVA that does not rely on a normality assumption. It tests whether the medians (rather than means) of k independent groups are all equal. If the result is found to be non-significant  and exact P-values are given in Additional file P-values provide precise information about whether two samples differ significantly in either means or medians. The procedure by which P-values are obtained incorporates information on the sample sizes and variability in both samples, making it unnecessary for the reader to extrapolate this information from error bars, which would require multiplying each standard error of the mean by a factor that depends on each sample size.Results are reported in the form of t-tests followed by the Hochberg procedure for multiple comparisons. For comparing relative SDS response indices, in addition to the t-tests described in Figure post hoc tests were performed to confirm statistical significance  Quantification of NLG-1 GRASP intensity (A) and neurite contact (B) for unc-7, ina-1, and sdn-1 mutants. (A) unc-7, ina-1, and sdn-1 have normal NLG-1 GRASP intensities, indicating that PHB-AVA synaptogenesis is not affected by the observed defects in PHB-AVA neurite contact or general nerve bundle fasciculation. Wild-type n = 216, unc-6 n = 87, unc-40 n = 85, unc-7 n = 39, ina-1 n = 41, and sdn-1 n = 41 animals. ***P < 0.001, NS, not significant, u-test. P-values were adjusted for multiple comparisons using the Hochberg method. (B) unc-7 mutants display defects in PHB-AVA neurite contact as severe as those in unc-6 and unc-40 mutants. ina-1 mutants display less severe neurite contact defects, and sdn-1 mutants do not have significant defects in neurite contact, although both mutants have previously characterized defects in general nerve bundle fasciculation. Wild-type n = 216, unc-6 n = 87, unc-40 n = 85, unc-7 n = 39, ina-1 n = 41, and sdn-1 n = 41 animals. ***P < 0.001, **P < 0.01, NS, not significant, t-test. P-values were adjusted for multiple comparisons using the Hochberg method.  Schematics,  micrographs of PHB-AVA NLG-1 GRASP signal,  cytosolic mCherry labeling PHB and AVA neurite contact, and  merged images. (C-F) Wild type, (G-J) unc-7, (K-N) ina-1, (O-R) sdn-1. Yellow scale bar: 2 μm.Click here for fileFigure S3 - in wild-type animals synapses occupy a fraction of the length of neurite contact between PHB and AVA. Quantification of the extent of PHB-AVA neurite overlap, and the sum of the lengths of all NLG-1 GRASP puncta in the preanal ganglion in wild-type animals. The sum of the lengths of all NLG-1 GRASP puncta indicates that only 22% of the neurite overlap region is occupied by PHB-AVA synapses. Wild-type n = 41 animals. ***P < 0.001, t-test. P-values were adjusted for multiple comparisons using the Hochberg method.Click here for fileunc-6 and unc-40-mediated cell migration and axon guidance pathway mutants exhibit no defects in SPRFigure S4 - . Quantification of (A) NLG-1 GRASP intensity and (B) neurite contact for age-1, clec-38, mig-10, unc-5, unc-115, and unc-129 mutants. (A) Axon guidance molecules age-1, clec-38, mig-10, unc-5, unc-115, and unc-129 have normal NLG-1 GRASP intensities, indicating that SPR is molecularly distinct from the classical unc-6 and unc-40-mediated axon guidance pathways. Wild-type n = 80- 127, age-1 n = 131, clec-38 n = 91, mig-10 n = 99, unc-5 n = 80, unc-115 n = 86, unc-129 n = 88 animals. NS, not significant, u-test. P-values were adjusted for multiple comparisons using the Hochberg method. (B) Axon guidance molecules age-1, clec-38, mig-10, unc-5, unc-115, and unc-129 have normal neurite contact, also indicating that the SPR pathway is molecularly distinct. Wild-type n = 80127, age-1 n = 131, clec-38 n = 91, mig-10 n = 99, unc-5 n = 80, unc-115 n = 86, unc-129 n = 88 animals. NS, not significant, t-test. P-values were adjusted for multiple comparisons using the Hochberg method.  Schematics,  micrographs of normal PHB-AVA NLG-1 GRASP signal,  cytosolic mCherry labeling normal PHB and AVA neurite contact, and  merged images. (C-F) Wild type, (G-J) age-1, (K-N) clec-38 (O-R) mig-10, (S-V) unc-5, (W-Z) unc-115, and (AA-DD) unc-129 animal. (EE-HH) unc-34 animals could not be assayed for SPR phenotypes due to penetrant neurite extension defects in PHB and sometimes in AVA. Yellow scale bar: 2 μm.Click here for fileunc-6 and unc-40 mutantsFigure S5 - active zone and postsynaptic components localize to the correct subcellular compartment in .  Wild type,  unc-6, and  unc-40 are labeled with the active zone markers syd-2::YFP (A-F) or GFP::elks-1 (G-L) expressed in PHB neurons or the postsynaptic marker nlg-1::YFP (M-R) in AVA. (A-L) Presynaptic active zone components are unaltered in unc-6 and unc-40 mutant animals, localizing to the distal region of the PHB axon within the preanal ganglion where PHB synapses normally form (boxed in white). Yellow scale bar: 2 μm. (M-R) Postsynaptic specializations also localize normally to the preanal ganglion (boxed in white) as well as along the ventral nerve cord where AVA receives synaptic input from other neurons. Yellow scale bar: 2 μm. (S) Quantification of pre- or postsynaptic marker fluorescence intensity using NIH ImageJ indicates no significant difference among wild type, unc-6, and unc-40 animals. For syd-2::YFP, wild type n = 3942, unc-6 n = 30, and unc-40 n = 29 animals. For GFP::elks-1, wild type n = 46, unc-6 n = 31, unc-40 n = 41. For nlg-1::YFP, wild type n = 40 to 42, unc-6 n = 36, unc-40 n = 43. NS, not significant, u-test. P-values were adjusted for multiple comparisons using the Hochberg method.Click here for fileFigure S6 - expression of UNC-6 in AVA and UNC-40 in PHB are sufficient for SPR.  Schematics and  micrographs of PHB-AVA NLG-1 GRASP signal,  cytosolic mCherry labeling PHB and AVA neurite contact, and  merged images. (A-D) Wild-type animal, (E-H) pAVA::unc-6 unc-6; animal, (I-L) pAVA::MTunc-6 unc-6; animal, (M-P) pPHB::unc-40 unc-40; animal, (Q-T) animal overexpressing pAVA::unc-6, (U-X) animal overexpressing pAVA::MTunc-6, (Y-BB) animal overexpressing pPHB::unc-40. (A-BB) Yellow scale bar: 2 μm. (CC) Schematic and (DD) micrograph of pPHB::unc-40::GFP in wild-type background. (EE) Schematic and (FF) micrograph of pPHB::unc-40::GFPunc-6; , indicating that localization of UNC-40 protein to the synaptic region of the PHB axon is not altered in the absence of its ligand UNC-6. (CC-FF) Yellow scale bar: 5 μm.Click here for fileunc-104 mutants have similar SPR defects to unc-6 mutantsFigure S7 - . Quantification of (A) NLG-1 GRASP intensity and (B) neurite contact in unc-104 mutants, which is required for UNC-6 localization. (A) unc-104 mutants have defects in NLG-1 GRASP intensity that are similar to those observed in unc-6 and unc-40 mutants. ***P < 0.001, NS, not significant, u-test. P-values were adjusted for multiple comparisons using the Hochberg method. (B) Defects in neurite contact are also similar in unc-104, unc-6, and unc-40 mutants. ***P < 0.001, NS, not significant, t-test. P-values were adjusted for multiple comparisons using the Hochberg method.  Wild-type n = 134, unc-6 n = 87, unc-40 n = 85, unc-104 n = 43 animals.Click here for fileMovie S1 - response of a wild-type animal to SDS. A wild-type animal is touched on the nose with a hair pick, eliciting backward motion. A dry drop of SDS is placed behind the animal. The animal quickly stops reversing when it encounters the SDS. The dry drop of SDS is outlined in red.Click here for fileunc-40 animal to SDSMovie S2 - response of an . An unc-40 mutant animal is touched on the nose with a hair pick, eliciting backward motion. A dry drop of SDS is placed behind the animal. The response time to SDS is longer than that displayed by wild-type animals. The dry drop of SDS is outlined in red.Click here for fileTable S1 - summary of statistical analysis. P-values for all statistical tests performed in this work.Click here for file