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# Gcode T0

0050 F1080 G1 X190. M104 T0 S200 ; start heating extruder 1 T0 ; set the active extruder to 0; settings from start_gcode G28 ; home all axes G1 Z5 F5000 ; lift nozzle; automatic settings after start_gcode M109 T0 S200 ; wait for extruder 1 to reach temperature T0 ; set the active extruder to 0 G90 ; use absolute coordinates G92 E0 ; reset the expected extruder. Use MathJax to format equations. print heads). How to convert an image into a 3D printable file. Upload a file to the selected location or create a new empty folder on it. 0*82 N7 G1 X2. Dual-Color Printing with Simplify3D Prepare separate processes to create stunning, multi-colored objects. When the extruder reaches. ; Final del gcode G91 ; posición relativa G1 Z10. * Ant run command line commands support. Here are some examples of the automatic G-Code: Before: ; automatic settings before start_gcode G21 ; set units to millimeters M107 ; fan off M190 S95 ; wait for bed temperature to be reached M104 T0 S210 ; start heating extruder 1 T0 ; set the active extruder to 0. G-Code comments begin at a semicolon, and end at the end of the line: T0 ; This is a comment G92 E0 ''';So is this''' G28. 67M132: Set PID D value 4. 74 Count X:11800 Y:12600 Z:4000. 00 F4800 G1 X-15. Tool Change G-CODE. Files for BFB/RapMan have the extension. Billing and Coding Guidelines. Some G words alter the state of the machine so that it changes from cutting straight lines to cutting arcs. ;end gcode M104 S0 T0 G162 Z G28 X Y M132 X Y Z A B G91 M18. That is a bitmap file, very large and mostly unusable, save the cad drawing as a. 0*82 N7 G1 X2. 2; automatic settings before start_gcode M104 T0 S215 ; start heating extruder 0 T0 ; set the active extruder to 0; settings from start_gcode. With any luck, I now know: What a given command does; What other interpreters do with that command. So, you want to request a 3D print job? Awesome! We've got some info on the process on our website that you should likely have a peek at first. Not exactly removal, but those settings should be derived from the values entered in Printer Settings->Machine limits in PrusaSlicer. The "G1 A10" won't work, only the E and switching with T are implemented. This in conjunction with --data-file and --fourier-option real allows to get the proper initial phase if t0's are known. Tool change. To connect, m ake sure the baud rate is set to 250,000 and that the USB port is the same as the one used on the IDE to compile the Arduino. 4 1 1 1 0 0 1200 0 0. The Custom G-code section of the Printer Settings tab has an option for inserting G-code between tool changes. Arduino BT w/ ATmega328P. 三菱ケミカルインフラテックの給水給湯用配管器具、耐候性保温材 ＜HON10-T0. A typical piece of GCode as sent to a RepRap machine might look like this: N3 T0*57 N4 G92 E0*67 N5 G28*22 N6 G1 F1500. 1f When a streamed g-code command throws a soft limit alarm (alarm:2) during a g-code stream, the sender properly stops the stream and displays a window s. G-Code Detailed Reference G commands G0/G1 - Linear Move. When in slicr you can designate which extruder but I am apparently missing something to make it work automatically select the proper extruder and it just goes to the T that is set in the g code. 1 wall_thickness = 0. At some point the the controller board failed. Hi I'm Michael, I love all things Science, Engineering, & 3D Printing. This solution article will explain how to set-up the printer as a 'Custom FFF printer' in Cura. In Marlin 1. here is my start gcode (copy and paste) to put in starting script ; -- START GCODE --M73 P0 (enable build progress) G21 (set units to mm) G90 (set positioning to absolute) G10 P1 X16. This works for the extruder as well. 05 0 0 1200 0 0. Use MathJax to format equations. org> Subject: Exported From Confluence MIME-Version: 1. However, the problem may need a bit more attention for the dryer to function properly. Don't wait hours for slicing that detailed model. Codes that are Relevant to the Dremel 3d45 3D printers at the GSD are:. G-code is the language of your 3d printer. Cura is a free slicer that can be used on your computer. Hi, See small section of the log file below FYI (from the Terminal window) in MatterControl 1. Several gcode settings will persist between prints if not reset, so I like to ensure everything is at defaults on completion. I will be glad to hear your opinion or advice. Even if your printer uses a different file format such as an. Quickly learn this code using our tutorial. G-code is a language in which people tell computerized machine tools how to make something. The Techno G-Code CNC Interface is designed to recognize some of the standard G/M codes. G20 and G21 G Code: Metric and Imperial Unit Conversion CNC Programming CNCCookbook’s G-Code Tutorial Introduction. It's better to strip these out on the host computer before sending the Gcode to your printer, as this saves bandwidth. At some point the the controller board failed. I have two questions about the tool change script. The below table shows the list of windows versions. The pin it self can not be used to run a fan as the output pins on Arduino's can't source more than a few milli amps. Smoothie reads G-code instructions, and converts those into movement, typically by turning motors. ) that are understood and interpreted by Mach3. 0, it seems to ignore these settings, and the sliced file always starts with:. ;end gcode M104 S0 T0 G162 Z G28 X Y M132 X Y Z A B G91 M18. That is a bitmap file, very large and mostly unusable, save the cad drawing as a. 25 5 5 5 1 1 0 0 0 0 50 120 0. I have tried manually adding an M206 X-10 line to the gcode file, just after G28 ; home all, however this has not had any effect: G90 M83 M106 S0 M140 S65 M190 S65 M104 S220 T0 M109 S220 T0 G28 ; home all M206 X-10 ; added by me G1 E-0. Tool change. jpg file and post it again, include all pertinent dimensions including the center positions of every radius in absolute coordinates, someone then may come along and help you without a tremendous amount of work. I just bought myself an i3 Mega and I'm running into an issue right out of the box. Windows Operating System Version Numbers. Posts: 417. Tn will change the tool to n for all future commands, it may appear anywhere on the line or on a line by itself T1 M200 M200 T1. If you just want to calibrate one of your extruder use “_T0” or “_T1” gcode. But bed temp is to low. 2; automatic settings before start_gcode M104 T0 S215 ; start heating extruder 0 T0 ; set the active extruder to 0; settings from start_gcode. G-Code is the most popular programming language used for programming CNC machinery. It's not in the same order of difficulty. 0*85 N8 G1 X3. 68M133: Set PID I limit value 4. This is the activation of tool head Zero. 0 share; Facebook; Twitter. csv = preswitchextruder. 85; G1 Z5 F5000 ; lift nozzle M109 S210 ; set the extruder temp and wait G28 X0 Y0 Z0 ; Home Z again in case there was filament on nozzle G29 ; probe the bed; automatic settings after start_gcode T0 ; set the active extruder to 0 G90 ; use. Cura is a free slicer that can be used on your computer. 200 F1002 ; layer 1, Z = 0. Therefore, the message you posted is NOT the message that the G code sender showed you. 00 F4800 G1 X-15. ICD-9 To ICD-10 Crosswalks (GEMs) ICD 9/10 GEMs (General Equivalency Mappings) For each ICD-9-CM code you can now view the ICD-10-CM equivalents and possibilities. Includes a list of all commands. G Code comments: N3 T0*57 ; This is a comment N4 G92 E0*67 ; So is this N5 G28*22 Comments and white space will be ignored by your RepRap Printer. 98 (set extruder speed) M103 T0 (Make sure extruder is off) M104 S225 T0 (set extruder temperature) M6 T0 (wait for toolhead parts, nozzle, HBP, etc. I don't feel like spending time and money on something that someone already has done. For example, for a Switch module, you can specify an arbitrary command in the 'input_on_command' and 'input_off_command'. There are no working codes right now. In the previous tutorial we have seen what G-code is, how it works and how you can write it by yourself on any text editor. N135 G01 X1. 0*33 シンボルと値の意味は、追って説明します。 g-codeがどのファームウェアに対応しているかを以下の表で示します。. Change those settings and you should get a reasonable set of M20x values in your gcode. Hi I'm Michael, I love all things Science, Engineering, & 3D Printing. Change those settings and you should get a reasonable set of M20x values in your gcode. print heads). gcode ****,M73 P100 ; end build progress,G1 Z150 F1000 ; send Z axis to bottom of machine,M140 S0 T0 ; cool down HBP,M104 S0 T0 ; cool down right extruder,M104 S0 T1 ; cool down left extruder,M127 ; stop blower fan,G162 X Y F3000 ; home XY maximum,M18 ; disable stepper,M72 P1 ; play Ta-Da song. Download this 3D slicer software from Cra. It is a series of commands that the machine understands and that determine where the toolhead moves, how fast, how much filament it extrudes and so on. 0 and up, the software endstops are adjusted to preserve the physical movement limits. This firmware determines what commands are valid, and which commands are not. Collez votre g-code dans la fenetre de gauche, vous pourrez visionner la simulation du parcours d'outil sur la droite. Note: Unless you are an expert on G-code, try not to write your own G-code because it may cause damage to your 3D printer. Machine code is the interface between software and hardware The processor is "hardwired" to implement machine code the bits of a machine instruction are direct inputs to the. You would then repead the process for the idle temperatures, but searching to "Tn Sfoo". * Fixed auto update for linux. The firmware does process the T0 on the M104 line, and sets the active temperature of tool 0 to the temperature specified. T0-T1: Select Tool; G-code Index. G-code (also RS-274), which has many variants, is the common name for the most widely used numerical control (NC) programming language. It was printing half off the edge of the bed. 0050 F1080 G1 X190. You can search on any combination of these fields. G-Code; Custom G-Code variables; All G-Code must be uppercase to function properly. Computerizedmachine-toolsareinstructedhowto. 5 board in conjunction with the Arduino DUE. 001 ) G20 G90 G64 G00 Z0. in text form Purely used for viewing. dxf to cnc converter free download. G-Code is the code that controls CNC machining centers, CNC lathes, CNC routers, and most 3D printers. A block is equivalent to a line of codes in a part program. The rest is pretty standard Prusa g-code. But no movement of any axis after I have done it. Message-ID: 1053571718. All these tools are defined and configured when the printer powers up with G-code commands, we will go over how exactly that works later. 0 T0 G0 X-110. ››More information from the unit converter. G-code stands for geometric code, where the variation of the alpha numeric patterns are communicating to the machine specific commands (N## G## X## Y## Z## F## S##). ;end gcode M104 S0 T0 G162 Z G28 X Y M132 X Y Z A B G91 M18. If you enter information into the "Registration", "Serial Number", "Aircraft Type/Name" fields or "ICAO Aircraft Type Designator" fields, you must enter at least two characters. The assumption may be that to change a file type, all you need to do is change the file's extension. gcode = replace. Collez votre g-code dans la fenetre de gauche, vous pourrez visionner la simulation du parcours d'outil sur la droite. Depending on your downloaded package you will find a "Z_calibration_0. Modify Start And End GCode After handwriting (modifying) code for pulley production I figured that there must be a way to have Skeinforge output the GCode with whatever you want at the start and end. The job of G-code-generating software is to turn a file created by a CAD (computer-aided design) program into G-code, which manages the motion of the device that is doing the making or modifying. Codes are small rewarding feature in Murder Mystery 2, similar to Promos, that allow players to enter a small portion of writing in their inventory and upon doing so, the player may receive a reward such as a knife, gun, or even a pet. For example, for a Switch module, you can specify an arbitrary command in the 'input_on_command' and 'input_off_command'. The "how" is defined by G-code instructions provided to a machine. This I put on the free fan slot of the RADDS boards. Make sure ever g code file you make has the save g code at the end. How To: Setup Grbl firmware, Drivers & Universal G-Code Sender Schematix. We give a quick definition of each g-code along with a link to tutorials and examples of how to use it. This includes the Jr line, the new Pro line and the wireless mini and unreleased micro. * Ant run command line commands support. Here is a Fanuc subprogram example which shows, how cnc machinists can make their cnc programs easier to write and maintain by useing sub-programming method. Joined: 1/29/2008. We will write some simple functions that will allow us to easily output G-code files. The standard for this general machine code, known as G/M-Code or, more commonly, G-Code, is EIA-274-D. I am using the RADDS 1. It is a series of commands that the machine understands and that determine where the toolhead moves, how fast, how much filament it extrudes and so on. Low cost printers with 3D printer software utilizing the fastest slicing algorithm. It is used mainly in computer-aided manufacturing to control automated machine tools. Default ABS Medium 0 0. It also takes you to the Haas factory floor to see how the code works on a real HAAS VF3 SS machining center. Even if your printer uses a different file format such as an. J'ai lu plusieurs posts de personnes ayant abandonne repetier/slic3r pour le logiciel Cura Au dire de ces personnes le tranchage est tres rapide par rapport a slic3 et cela je le confirme, sur plusieurs de mes stl le tranchage n'a pris que quelques secondes au lieu de plusieurs minutes sous slic3r. Select the process you'd like to assign to the printer's left extruder (T0), then click "Edit Process Settings” in the lower left-hand corner. The Duet Maestro control board runs RepRap Firmware. com。【5,000円（税別）以上のご注文で送料無料】【平日16時までのご注文は最短即日出荷】. 2; firstLayerThickness = 0. In Marlin 1. Options: Use 0x and comma as separator (C-like) Insert newlines after each 16B. Make sure ever g code file you make has the save g code at the end. ; **** Replicator 2X end. 00 Maximum Acceleration (units/s2): M201 X1000 Y1000 Z100 E10000 Acceleration (units/s2): P. M190 ;wait until bed has heated M117 Heating Nozzles M104 T0 S{print_temperature} ;Set target temperature for the nozzle 1. The temperature that I have set for both extruders is 245 yet when my Start GCode calls for M104 {material_print_temerature} T0/T1 it sets it to 210. UArm gCode is an important part of the uArm firmware 2. General Motors Engines We’ve compiled a comprehensive guide of General Motors engines used around the world. Absolute Positioning. Joined: 1/29/2008. Tool Offset Variables - FS 10/11/15/16/18/21 for Turning 158 Tool Setting 158 Assignments for 64 Offsets or Less - Memory Type A 159. Notice the comment in the input box:. 95 kB - downloaded 30 times. (Slicing software will do this automatically). Here are some examples of the automatic G-Code: Before: ; automatic settings before start_gcode G21 ; set units to millimeters M107 ; fan off M190 S95 ; wait for bed temperature to be reached M104 T0 S210 ; start heating extruder 1 T0 ; set the active extruder to 0. Parameters Description Example G0 Axis [X/Y/Z] Position Rapid Movement G0 X50 G1 Axis [X/Y/Z/E] Position Feed [F] Controlled Movement G1 F150 X10 G4 Time in ms [P] Dwell / Wait G4 P500 G20 none Set units to inch G20 G21 none Set units to mm G21 G28 Home G28 X Y G90 none Absolute Positioning G90. Semicolons allow you to write comments in the g-code. G-code is the generic name for a plain-text language that CNC machines can understand. To calculate thedrill revolutions per minute (RPM) from the surface speeds, the following formulae should be used, where D=diameter of drill. Based on the standard gCode protocol, we add a new protocol head in front of the gCode so that it can be more easily to use and debug. Head Drivers 110 3 8 Metric Hex Head Drivers 3452S55 T0-55 225060200 103. This should work with just about any printer but I've only tested it with Ender 3 so far. A typical piece of G-code as sent to a RepRap machine might look like this: N3 T0*57 N4 G92 E0*67 N5 G28*22 N6 G1 F1500. Provide details and share your research! But avoid … Asking for help, clarification, or responding to other answers. 8 fill_density = 5 print_speed = 30 print_temperature = 230 print_temperature2. FlashForge Creator Pro (modified) PLA High Both Extruders 0 0. Re: Root Profile for Cura by ModernGnome on Tue Oct 20, 2015 11:42 am No, WTK is a binary output only file that is, as far as I can see, identical to X3G (but with heatbed information). Hi, Im wondering if anyone manage to have a perfect setup to have the MakerBot rep 2 work correctly with the Makerbot Rep2. G21 ; Units in mm M149 C ; Units in Celsius Filament settings: Disabled M200 D1. How to send 3D Printer g-codes to the. Go to Print » Print from local file » Calibration files. Traditionally, this will consist of homing the printer, potentially purging the nozzle, wiping the nozzle and running an auto-leveling function if the printer supports it. parameters. -) A pause command that I could insert into the g-code that would cause the print to pause (if printing to USB) so I could do things like add an item into the print or change the colour. Left is logical tool T0 and right is logical tool T1. 00 at line 12. A printer with more than one extruder can be used in different ways. 00 T0 M140 S0 M104 S210 T0 M104 S0 T1 M107 G90 G28 M132 X Y Z A B G1 Z50. 099 F4800 G1 E0. All these tools are defined and configured when the printer powers up with G-code commands, we will go over how exactly that works later. Need help to convert *. G-Code Description Example ; G0: Move to the given coordinates. (We offer the code of decode the standard gCode) 1. How do I login to GCodes? Before you can login to GCodes, you must add your first GCode to wallet (redeem) and register an account. M06 is the M-code Used in the CNC Program to activate the automatic tool change along which T number called on the program. They look something like this: M109 S110 T0 (set HBP temperature) M104 S210 T0 (set extruder temperature) (temp updated by printOMatic). 0 G161 Z F100 M132 X Y Z A B (**** end homing ****) G1 X-110. Slic3r G-code for dual extrusion printing on the Flashforge Dreamer In this article the complete configuration of Slic3r for use with the Flashforge Dreamer is discussed. Examples M503; Print detailed settings M503 S0; Print shorted gcode settings. There are no working codes right now. [email protected] In regards to 3D printing, g-code contains a series of commands that move parts of the printer. Includes a list of all commands. Could this be done via gcode commands at the end of the print Job?. Multi-award-winning products with different sizes and options. gcode file to Chroma - GCode file - Chroma Profile Turns out the problem was a T0 in the start gcode. 3697 Z-1 R2 M7 G4 P. -) A g-code editor built in so I could make small changes to the code before printing. * More informations on gcodes: last print time, remember materials and store a note. If you have a multi-part model, that defines distinct regions, you can assign each one of them to an extruder. Tool change. GitHub Gist: instantly share code, notes, and snippets. Maîtriser le GCODE est essentiel à partir d'un certain niveau d'utilisation pour améliorer les process d'impression et les adapter vraiment à son application. 0*85 N8 G1 X3. Please not that this is the first G code I have seen that is like this “ M8012 I200” in this case the M8012 is the code then “ I” is the actual settings your adjusting so in the above example it is 200. test routers demos lorem install operating system WOOD ROUTER Specialty CNC beam punch laser laser additive router gcode g-code m-codes mcode mill retrofit lathe Testimonials control Controller X15-250 IMTS-2016 imts Plasma THC Bender Tube Chiyoda TECO Water Jet Grinder Apollo 1000 Series oxy fuel control kits TECHSOLVE HYBRID MANUFACTURING. Set the T0 temperature to Active and allow the Hotend to heat up. build volume. What is G-code? INTRODUCTION. G-code is a language in which people tell computerized machine tools how to make something. Replace Monoprice Select Mini Controller With Arduino Mega & Ramps 1. 11 - First Print. Movement defaults to using absolute coordinates. You would then repead the process for the idle temperatures, but searching to "Tn Sfoo". 0*33 The meaning of all those symbols and numbers (and more) is explained below. and the big obvious was that it was missing 4 lines of code in the gcode example. Finally, T0 always means the first head on the first yoke, regardless of how or why we are referring to it. 67M132: Set PID D value 4. print heads). 25 5 5 5 1 1 0 0 0 0 50 120 0. Could this be done via gcode commands at the end of the print Job?. JPG) to 3D STL files! Use this free and fast online tool to convert your. 0337 G1 X191. Search for aircraft on the G-INFO database. This course teaches the fundamentals of G-Code to the programmers, engineers, and designers that need to write, read, and edit it. This section is still under development. Chart of Addresses. RepRapFirmware supports multiple tools (i. M190 ;wait until bed has heated M117 Heating Nozzles M104 T0 S{print_temperature} ;Set target temperature for the nozzle 1. 200 F1002 ; layer 1, Z = 0. Disclaimer: Het gebruik van software, downloads, scripts en uitleg op deze website is geheel op eigen risico en is bestemd voor educatief gebruik, DomoticX is niet aansprakelijk voor de schade die, rechtstreeks of onrechtstreeks het gevolg is van gebruik van deze website!. G90 Usa coordinate assolute – (g90 g code oppure gcode g90). When you are happy with the Hotend and plastic is extruding set the T0 temperature to Standby. Smoothie reads G-code instructions, and converts those into movement, typically by turning motors. While that might sound pretty trivial to do, the laws of physics actually make this a bit more challenging that one might expect. CPT® Code 90632 for Medicine Services and Procedures and more details about Vaccines, Toxoids. 0 T0 G0 X-110. Precision printing, efficient cost. PK rfj3 META-INF/PK rfj3m¾ÚÖGG META-INF/MANIFEST. Standard Marlin compatible G-codes can be found at the RepRap wiki: G-code. For the following G-code “M48 P50 X100 Y100 V4” was used to obtain the values for the table above. 5 Y-74 Z150 F3300. 04) single right. Raise3D's Pro2 has dual extrusion capabilities, a sizable 12×12×11. Once you have the correct cabling, all you have to do is match the baud rate and you are off and runningwell, mostly! Here are the steps to your success:. 85; G1 Z5 F5000 ; lift nozzle M109 S210 ; set the extruder temp and wait G28 X0 Y0 Z0 ; Home Z again in case there was filament on nozzle G29 ; probe the bed; automatic settings after start_gcode T0 ; set the active extruder to 0 G90 ; use. This includes the [previous_extruder] and [next_extruder] variables. 5 E-5 X-20 Y. Basically because as each vendor has a problem to solve it shows up as a new gcode. Connect to the E3 or E4 to your computer using your USB cable. A list of g-codes and m-codes for milling in the Fanuc, LinuxCNC, GRBL, and Haas dialects. com/watch?v=UWysgnGZcU4&list=PLyaleIbkr. With any luck, I now know: What a given command does; What other interpreters do with that command. ; -- start of END GCODE – G92 Z0 G1 Z10 F400 M18 M109 S0 T0 M104 S0 T0 M73 P100 (end build progress) G162 X Y F3000 M18 ; -- end of END GCODE – Enable plugins Enter correct path in “cura_x3g path”. However, the problem may need a bit more attention for the dryer to function properly. An end g-code snippet concludes the print and typically contains housekeeping functions like parking the head to the side and cooling down the extruder. The Duet Maestro control board runs RepRap Firmware. If you have a single part model, you can assign different roles to each extruder: for example, you can infill using a larger nozzle or you can build support material with soluble filament. The CAD/CAM software and the machine controller will take care of all of this for you. On a Fanuc control it's a G code usually G54 although as standard you have six of these. Previous step. It is used mainly in computer-aided manufacturing to control automated machine tools. G28 gives me this. Smoothie reads G-code instructions, and converts those into movement, typically by turning motors. 5 and we've been busy updating new github. 0*82 N7 G1 X2. A transaction code is used to access functions or running programs (including executing ABAP code) in the SAP application more rapidly. 5 board in conjunction with the Arduino DUE. Excellon file is a text-based file format which is used to control the actions of a CNC drilling machine, commonly used in the drilling of printed circuit boards (PCB). M104 T0 S200 ; start heating extruder 1 T0 ; set the active extruder to 0; settings from start_gcode G28 ; home all axes G1 Z5 F5000 ; lift nozzle; automatic settings after start_gcode M109 T0 S200 ; wait for extruder 1 to reach temperature T0 ; set the active extruder to 0 G90 ; use absolute coordinates G92 E0 ; reset the expected extruder. gcode” in the folder. dxf to cnc converter free download. DRL file is an Excellon Drill File. org> Subject: Exported From Confluence MIME-Version: 1. Basically because as each vendor has a problem to solve it shows up as a new gcode. 00 F400 G161 X Y F3300 M6 T0 M907 X100 Y100 Z40 A80 B20 M108 T0 G1 Z. T0-T1: Select Tool; G-code Index. Line 5 retracts 2mm of filament (E-2). M190 ;wait until bed has heated M117 Heating Nozzles M104 T0 S{print_temperature} ;Set target temperature for the nozzle 1. The following code works smoothly: G1 X0 Y0 Z5 F1000 ; lift nozzle <- M127 M104 S205 T0 ; set temperature. Absolute Positioning. 6, I had my CuraEngine slicer settings set to add custom start and end gcode (to do a hotend purge, set up for volumetric extrusion, etc). However, the problem may need a bit more attention for the dryer to function properly. Home CNC gcode grbl robot How to use GRBL for CNC control. (Slicing software will do this automatically). 0 G161 Z F100 M132 X Y Z A B (**** end homing ****) G1 X-110. 0-beta motion G4 - Dwell. in order to control the second extruder. G54~G59 - Workpiece coordinate system settings #1 t0 #6; Mill and Lathe G61 - Exact stop check; Mill and Lathe G65 - Custom macro call; Mill and Lathe G70 - Finish cycle; Lathe G71 - Rough turning cycle; Lathe G72 - Rough facing cycle; Lathe G73 - Irregular rough turning cycle; Lathe G73 - Chip break drilling cycle; Mill G74 - Left hand tapping. stl (foot shape) file exported from a 3D scanner and I need to convert it in to a solid shape to use it in Ansys. It is used mainly in computer-aided manufacturing to control automated machine tools. Material removal is simulated in real-time,. Machine code is the interface between software and hardware The processor is "hardwired" to implement machine code the bits of a machine instruction are direct inputs to the. A search function and a replace function would be a requirement in it. G1 is a normal Gcode command and is used for controlled movement in a straight line. 6_(T0 and/or T1). 0*82 N7 G1 X2. Start G-Code for dual extruder. Re: Root Profile for Cura by ModernGnome on Tue Oct 20, 2015 11:42 am No, WTK is a binary output only file that is, as far as I can see, identical to X3G (but with heatbed information). * Edit gcode after uploading. 2 wall_thickness = 0. Tool change. 4848 Basic Concept of PartBasic Concept of Part ProgrammingProgramming Preparatory command (G code) The G codes prepare the MCU for a given operation, typically involving a cutter motion. Previous step. For calibration both extruder extruders use “_T0_T1” gcode. Sin embargo, x3g es un lenguaje binario (basado en unos y ceros) más difícil de entender que Gcode. For example, for a tool change in the first instance and at the end of the job in the second. This gcode will raise the extruder before heating up to prevent the extruder from melting a hole int he bed at the start point. If not, go back into Z offset calibration and make minor adjustments based on your test print results using the pictures below as a reference:. However, the problem may need a bit more attention for the dryer to function properly. I flashed “MPCNC511_GLCD_T8″(June 2017) and I also have a 100k resistor on T0. If you try to copy this gcode for use with a dual. The T With the release of Repetrel v5, we are happy to announce that the means of addressing heads for T Commands and T Variables within M Commands has been unified. Craftunique is a 3D printer manufacturer and software developer company. With the above, you would then open the resulting gcode in a text editor, and do a search/replace for, for instance, "T1 Sbar" and replace all instances with "T1 S245"; then you'd also search for "T0 Sbar" and replace all instances with "T0 S220". With any luck, I now know: What a given command does; What other interpreters do with that command. Upload a file to the selected location or create a new empty folder on it. See the G-code page for information about software to use and how to send G-code. 4 is Open Hardware, the entire schematic and pinout is available or download diagram with pinout as one image (same as on the side) – but it’s also a hassle to plug correctly as the board plug descriptions are. * Fixed g-code pagination. Загрузка 28. This page explains how to configure the different motion control parameters you can tune in Smoothie. In the Start G-Code box, remove the text that is currently there and enter the following: M118 X15. The drivers are set to around 0. 04 (big changes post 15. G29 gives me this - yes it's bad but i have a new build plate ordered! Bed Level Correction Matrix: +0. Re: Simplify 3D, G-Code changes, questions & answers Post by Tim » Tue Oct 06, 2015 8:53 pm Jules wrote: The thread usually falls off before it hits the bed, and by the time the right nozzle reaches the bed, the movement of the print head after the left nozzle wipe takes the right nozzle off at an angle towards the center, so it doesn't follow. The firmware does process the T0 on the M104 line, and sets the active temperature of tool 0 to the temperature specified. The FlashForge Creator family of 3D printers are great printers for the price, but over the past several months, I have had to solve a number of problems and research a lot of answers in order to get everything working as I wanted. Using a modern-day desktop CNC machine and software, you’ll never have to enter G-code manually, unless you want to. Working Subscribe Subscribed Unsubscribe 27. Raise3D's Pro2 has dual extrusion capabilities, a sizable 12×12×11. Hi, See small section of the log file below FYI (from the Terminal window) in MatterControl 1. On a Fanuc control it's a G code usually G54 although as standard you have six of these. 0 T0 G0 X-110. M140 S[bed0_temperature] T0 ; Start heating bed M190 S[bed0_temperature] T0 ; Heat bed and wait for temperature to stabilize M104 S[extruder0_temperature] T0 ; Start heating right extruder M109 S[extruder0_temperature] T0 ; Heat right extruder. If not, go back into Z offset calibration and make minor adjustments based on your test print results using the pictures below as a reference:. Some G words alter the state of the machine so that it changes from cutting straight lines to cutting arcs. Once you have finished creating toolpaths, select the operations or the setups that you. 5 and we've been busy updating new github. A file containing RepRap G-code usually has the extension. G21 ; Units in mm M149 C ; Units in Celsius Filament settings: Disabled M200 D1. T16 as individual ‘spray tools’, and use the P Q R parameter variables to define up to 72 bits of nozzle control bitmap, and the S parameter to control the. Traditionally, this will consist of homing the printer, potentially purging the nozzle, wiping the nozzle and running an auto-leveling function if the printer supports it. JPG 2D images into 3D. G29 gives me this - yes it's bad but i have a new build plate ordered! Bed Level Correction Matrix: +0. 0] Shows the GRBL version and source code build date. Convert 2D images to 3D objects Convert 2D pictures and drawings into 3D STL (Stereolithography) files suitable for 3D printing or CNC routing. All these tools are defined and configured when the printer powers up with G-code commands, we will go over how exactly that works later. To calculate thedrill revolutions per minute (RPM) from the surface speeds, the following formulae should be used, where D=diameter of drill. For the 2nd Ashtar K 3D Printer I used (2018/11) RAMPS 1. What is G-code? INTRODUCTION. 00 F4800;preExtrude:0. Default MDC. 8 fill_density = 5 print_speed = 30 print_temperature = 230 print_temperature2. Very happy with the performance of the new version of Cura 4. Telehealth and Telemedicine Policy Page 1 of 6 UnitedHealthcare Oxford Reimbursement Policy Effective 05/01/2020 ©1996-2020, Oxford Health Plans, LLC TELEHEALTH AND TELEMEDICINE POLICY Policy Number: ADMINISTRATIVE 114. The problem now is that when I used this g code in the firmware the steppers stop working. 0*33 G-code can also be stored in files on SD cards. csv = preswitchextruder. UArm gCode is an important part of the uArm firmware 2. Thus you could use G92 to set the middle of the bed to 0,0 and then run. The T With the release of Repetrel v5, we are happy to announce that the means of addressing heads for T Commands and T Variables within M Commands has been unified. It is customary to use T0 on a random toolchanger machine to track an empty pocket, so that it behaves like a nonrandom toolchanger machine and. To use a variable, type the G-code as normal, but replace the value with a variable in square brackets[]. Here are some examples of the automatic G-Code: Before: ; automatic settings before start_gcode G21 ; set units to millimeters M107 ; fan off M190 S95 ; wait for bed temperature to be reached M104 T0 S210 ; start heating extruder 1 T0 ; set the active extruder to 0. Hi, I would like to make the Printer retract the material after print Job in order to not clog the nozzle and to make filament changes easier. Billing and Coding Guidelines for MS-004, Bone Mass Measurement. Joined: 1/29/2008. I have tried to add all the OS version numbers, if I have missed any, do write in the comments. com | Replacement Parts for Fadal | Fadal CNC Machines. gcode **** M73 P100 ; end build progress G1 Z155 F1000 ; send Z axis to bottom of machine M140 S0 T0 ; cool down HBP M104 S0 T0 ; cool down right extruder. This page explains how to configure the different motion control parameters you can tune in Smoothie. Best way to convert your DXF to SVG file in seconds. The Duet Maestro control board runs RepRap Firmware. ICD-9 To ICD-10 Crosswalks (GEMs) ICD 9/10 GEMs (General Equivalency Mappings) For each ICD-9-CM code you can now view the ICD-10-CM equivalents and possibilities. これを、FlashPrint にドロップすれば、思惑通りの動きがシミュレートされます. Find 14 used 1964 Ford Fairlane as low as \$20,000 on Carsforsale. M104 T0 S200 ; start heating extruder 1 T0 ; set the active extruder to 0; settings from start_gcode G28 ; home all axes G1 Z5 F5000 ; lift nozzle; automatic settings after start_gcode M109 T0 S200 ; wait for extruder 1 to reach temperature T0 ; set the active extruder to 0 G90 ; use absolute coordinates G92 E0 ; reset the expected extruder. 5 1 1 0 0 1200 0 0. How to send 3D Printer g-codes to the. 001 ) G20 G90 G64 G00 Z0. gcode G90 M82 M106 S0 M104 S210 T0 M109 S210 T0 G28 ; home all axes G92 E0 G1 E-3. You would then repead the process for the idle temperatures, but searching to "Tn Sfoo". In the Start G-Code box, remove the text that is currently there and enter the following: M118 X15. 2 M9 Fist of all you omit arguments and you use M codes on same line so more valid combination must be looks something like this G81 X-21. Now, with RepetierHost 2. What is G-code? INTRODUCTION. I have tried to add all the OS version numbers, if I have missed any, do write in the comments. 1 G1 X0 Z0 G05 X1. 5 Y-74 Z150 F3300. G-code Appeared in 1950s (first edition)Designed by Massachusetts Institute of Technology Major implementations many, mainly Siemens Sinumeric, FANUC, Haas, Heidenhain, Mazak. installation de GRBL sur un ARDUINO UNO GRBL Installation on an ARDUINO UNO Board CNC DIY chanel: https://www. At some point the the controller board failed. It is used mainly in computer-aided manufacturing to control automated machine tools. Running a Anilam 4200T Setup, however I just can't seem to get the G code down. gcode M117 Heating Bed M106 S255 ;set fan to max M140 S{print_bed_temperature} ;Set target temperature for the heated bed. Пример: M218 T0 X50 Y10. T0 ; set the active extruder to 0; settings from start_gcode G28 X0 Y0 Z0 ; home all axes M565 Z-0. My prints always start with T0 then a change top T1 is made. The Smoothieware configuration file permits the association of additional codes with some tool functions. 20% (VAT on Income) This is the standard sales tax rate for a sale to a VAT registered business. The following code works smoothly: G1 X0 Y0 Z5 F1000 ; lift nozzle <- M127 M104 S205 T0 ; set temperature. Beispiele für G0 und G01. This should work with just about any printer but I've only tested it with Ender 3 so far. Now, with RepetierHost 2. I looked at the gcode chroma is putting out under the side transition option. Here’s my current end g-code:. (Slicing software will do this automatically). For calibration both extruder extruders use “_T0_T1” gcode. G31, M564 and T0 are all valid G-code commands as well. Hi, Im wondering if anyone manage to have a perfect setup to have the MakerBot rep 2 work correctly with the Makerbot Rep2. Extrusion rate (M221) is set to 100%. Material removal is simulated in real-time,. 6, I had my CuraEngine slicer settings set to add custom start and end gcode (to do a hotend purge, set up for volumetric extrusion, etc). I would like to switch on an LED when changing from extruder 0 (main material) to extruder 1 (support). Set the T0 temperature to Active and allow the Hotend to heat up. 0 T0 G0 X-110. The HCPCS codes range Transportation Services T2001-T2007 is a standardized code set necessary for Medicare and other health insurance providers to provide healthcare claims. 0337 G1 X191. The following code works smoothly: G1 X0 Y0 Z5 F1000 ; lift nozzle <- M127 M104 S205 T0 ; set temperature. Fast G-code generation is fast. Want to help…. with the users temps of course. -) A pause command that I could insert into the g-code that would cause the print to pause (if printing to USB) so I could do things like add an item into the print or change the colour. Introducción. Modify Start And End GCode After handwriting (modifying) code for pulley production I figured that there must be a way to have Skeinforge output the GCode with whatever you want at the start and end. Notice the comment in the input box:. 1 - includes detailed rules, notes, synonyms, ICD-9-CM conversion, index and annotation crosswalks, DRG grouping and more. 0 F7200; Link vom Bett positionieren, Position für das Aufheizen einnehmen G1 Z3. 5 Y-74 G0 Z0. UArm gCode is an important part of the uArm firmware 2. G20 geändert werden. {IF NEWTOOL=0}M133 T0; Stabilise T0 temperature {IF NEWTOOL=1}; LEFT Extruder is active {IF NEWTOOL=1}M104 S165 T0; Cool inactive extruder to 165c (T0) {IF NEWTOOL=1}M104 S210 T1. Finite Element Method. On random tool changer machines, G41/G42 D0 applies the TLO of the tool T0 defined in the tool table file The G81 Example 1 demonstrates how a canned cycle could be used to produce 8 holes with ten lines of G code within the canned cycle mode. Because the code can only turn on OR. G-Code: simulateur de parcours d'outil. Codes are mostly always given away at Nikilis's twitter page. T15 (so while E0 is always 0%/100% mix then T0 can be overridden to for example 30/70), so in gcode when you use one of these assigned tools you get a different mix. Go to Print » Print from local file » Calibration files. Posted November 6, 2012 · G-Code to set 2nd Extruder Temp Cura already has dual-extrusion support for 2 versions now. -) A pause command that I could insert into the g-code that would cause the print to pause (if printing to USB) so I could do things like add an item into the print or change the colour. Could this be done via gcode commands at the end of the print Job?. G-Code comments begin at a semicolon, and end at the end of the line: T0 ; This is a comment G92 E0 ''';So is this''' G28. G-code is a language in which people tell computerized machine tools how to make something. The rest is pretty standard Prusa g-code. 5 and possible newer. ;start gcode M118 X15. Return to G-code list G94 Units Per Minute In units per minute feed rate mode, an F word on the line is interpreted to mean the controlled point should move at a certain number of inches per minute, millimetres per minute, or degrees per minute, depending upon what length units are being used and which axis or axes are moving. 1f When a streamed g-code command throws a soft limit alarm (alarm:2) during a g-code stream, the sender properly stops the stream and displays a window s. Using a modern-day desktop CNC machine and software, you’ll never have to enter G-code manually, unless you want to. DRL file is an Excellon Drill File. 0-beta motion G0, G1 - Linear Move. When I last sliced a dual extrusion model, slic3r did generate separate T0 and T1 commands in the gcode, as well as putting the T0 and T1 parameters on the M104 and M109 commands. Even if your printer uses a different file format such as an. Don't wait hours for slicing that detailed model. I just upgraded to Simplify3D version 3. [email protected] 0, it seems to ignore these settings, and the sliced file always starts with:. 100% free, secure and easy to use! Convertio — advanced online tool that solving any problems with any files. M104 S[first_layer_temperature_0] T0; set 1st nozzle heater to first layer temperature. T0 ; set the active extruder to 0; settings from start_gcode G28 X0 Y0 Z0 ; home all axes M565 Z-0. Here is a G code example which shows multiple ways for Arc programming on Fanuc CNC lathe machines. This gcode will raise the extruder before heating up to prevent the extruder from melting a hole int he bed at the start point. In fundamental terms, G-code is a language in which people tell computerized machine tools what to make and how to make it. Typically, the values not set up by the CAM program within the G-code are set up by the Post Processor for each machine type. 0*82 N7 G1 X2. Introducción. G-Code to be run after every tool change (T0, T1, T2, etc. This file calls multiple machine files listed above with the M98 command. JPG) to 3D STL files! Use this free and fast online tool to convert your. The problem now is that when I used this g code in the firmware the steppers stop working. 0*33 The meaning of all those symbols and numbers (and more) is explained below. Word shows you, in list format, how the selected text is formatted with respect to the Font, Paragraph, and Section. In RepetierHost 1. com。【5,000円（税別）以上のご注文で送料無料】【平日16時までのご注文は最短即日出荷】. 2 Walls: 1. Once you have the correct cabling, all you have to do is match the baud rate and you are off and runningwell, mostly! Here are the steps to your success:. Cura 3 - recent full release sets wrong temperature for material. gcode is to set the temperature 10C higher than I want with M109 to give the tip a chance to catch up before the print starts. -) A pause command that I could insert into the g-code that would cause the print to pause (if printing to USB) so I could do things like add an item into the print or change the colour. PK rfj3 META-INF/PK rfj3m¾ÚÖGG META-INF/MANIFEST. With the release of Repetrel v5, we are happy to announce that the means of addressing heads for T Commands and T Variables within M Commands has been unified. Example - [G0 G54 G17 G21 G90 G94 M0 M5 M9 T0 S0. 1588577015317. The example below is for calibrating both extruders. ) that are understood and interpreted by Mach3. How many revolutions per minute in 1 RPM? The answer is 1. Therefore, the message you posted is NOT the message that the G code sender showed you. Generally there is one international standard - ISO 6983. M140 S[bed0_temperature] T0 ; Start heating bed M190 S[bed0_temperature] T0 ; Heat bed and wait for temperature to stabilize M104 S[extruder0_temperature] T0 ; Start heating right extruder M109 S[extruder0_temperature] T0 ; Heat right extruder. Arduino BT w/ ATmega328P. However, CuraEngine does not handle E value (extruder step) correctly. Le GCODE c'est la vie ! Toutes les imprimantes 3D du commerce fonctionnent en GCODE, ainsi que de nombreux équipements de fabrication numérique (fraiseuses, tours numériques, etc). I have tried manually adding an M206 X-10 line to the gcode file, just after G28 ; home all, however this has not had any effect: G90 M83 M106 S0 M140 S65 M190 S65 M104 S220 T0 M109 S220 T0 G28 ; home all M206 X-10 ; added by me G1 E-0. Navigate to the "Auto-Configure Extruders" section in the upper right-hand corner of your process settings, then select ”Dual Mode - Left Extruder (T0)” — this will assign all process settings to. : G21 (set units to mm) G90 (set positioning to absolute) M108 R3. Mach3 g code language reference 1. 0*82 N7 G1 X2. -) A pause command that I could insert into the g-code that would cause the print to pause (if printing to USB) so I could do things like add an item into the print or change the colour. In fundamental terms, G-code is a language in which people tell computerized machine tools what to make and how to make it. I'll warn that it's a pretty popular service and it takes quite a while to process all the requests, get them printed, etc. year for two different flu seasons and the provider would be reimbursed for each. Maîtriser le GCODE est essentiel à partir d'un certain niveau d'utilisation pour améliorer les process d'impression et les adapter vraiment à son application. As with all custom G-code sections, placeholder variables can be used to reference Slic3r settings. Hi, I would like to make the Printer retract the material after print Job in order to not clog the nozzle and to make filament changes easier. gcode M117 Heating Bed M106 S255 ;set fan to max M140 S{print_bed_temperature} ;Set target temperature for the heated bed. RepRap GCode Cheat Sheet Comm. 0, it seems to ignore these settings, and the sliced file always starts with:. NEED TO CONVERT YOUR DESIGNS FOR CAD? Scan2CAD is the market leading conversion software for CAD. Download this 3D slicer software from Cra. However, CuraEngine does not handle E value (extruder step) correctly. Find-A-Code also includes the FULL ICD-10-CM and ICD-10-PCS codes sets. The below table shows the list of windows versions. com/watch?v=UWysgnGZcU4&list=PLyaleIbkr. STL (stereolithography) files suitable for printing with a 3D printer or for loading into your favourite 3D editing package. M140 S60 T0 M134 T0 ; stabilize bed temperature M104 S235 T0. 0 T 1 (set extruder speed left). The Advance Design era (1947-1955) has been overlooked for long enough and Deve Krehbiel, a well-known restoration expert from DevesTechnet. Hi Isaac, the CoolEn is an 5v output pin that goes when the Coolant G-Code command is entered. Problems with gcode when changing tool on dual Discussion in ' Calibration, Help, and Troubleshooting ' started by Alex9779 , Mar 24, 2016. 0*33 G-code can also be stored in files on SD cards. 0 ) T0 M6 ( Profile1 ) G17 M3 S1000. Topic: print gcode files to miniMaker I made a new utility called miniMover that lets you send gcode files to a miniMaker, or any other XYZ 3D printer that uses the V3 serial protocol. ) PK jfj3 javax/jnlp/PK ifj3 ó0 javax/jnlp. The first stop on the 5-Step G-Code Coordinate Pipeline is Unit Conversion, and this chapter is all about delving into that subject. Wisconsin Physicians Service Insurance Corporation. 0*85 N8 G1 X3. In this post we will see how they perform very easily the gcode with Repetier Host, and so get: - An awesome Gcode viewer, in real time. The starting script from S35 is what pretty much unchanged from the original S3D profile; just a little PID tuning in there for bed and tool. 0050 F1080 G1 X190. Set the T0 temperature to Active and allow the Hotend to heat up. Hi, See small section of the log file below FYI (from the Terminal window) in MatterControl 1. In the below table if you look carefully at the operating system version number it is made of up Major Version, Minor Version and Build number. a RS-274), which has many variants, is the common name for the most widely used numerical control (NC) programming language. G and M-code Reference Using Mach3Mill Rev 1. Subscribe to AAPC Coder and get the code details in a flash. The extruder activate_gcode and deactivate_gcode config options have been removed. Collez votre g-code dans la fenetre de gauche, vous pourrez visionner la simulation du parcours d'outil sur la droite. The Duet Maestro control board runs RepRap Firmware. Traditionally, this will consist of homing the printer, potentially purging the nozzle, wiping the nozzle and running an auto-leveling function if the printer supports it. I contacted Maylan the original manufacturer of the board. Introducción. Plan B code is based on Gcode, it shares much of the structure of Gcode, but it has a few functions added specifically for Plan B. and the big obvious was that it was missing 4 lines of code in the gcode example. A G-Code file provides a complete set of machine instructions which the printer needs to complete a part. Codes are mostly always given away at Nikilis's twitter page. 135 T0 ; tool H0. G-Code Commands. 1f When a streamed g-code command throws a soft limit alarm (alarm:2) during a g-code stream, the sender properly stops the stream and displays a window s. Hi, See small section of the log file below FYI (from the Terminal window) in MatterControl 1. 2016 2017 2018 2019 2020 Billable/Specific Code. Hi, I would like to make the Printer retract the material after print Job in order to not clog the nozzle and to make filament changes easier. Return to G-code list G94 Units Per Minute In units per minute feed rate mode, an F word on the line is interpreted to mean the controlled point should move at a certain number of inches per minute, millimetres per minute, or degrees per minute, depending upon what length units are being used and which axis or axes are moving. 0*82 N7 G1 X2. G20 imperial units (inches for positions and inches per min for feedrates) G21 metric units (mm and mm per min) G54 Fixture offsets. Hi I'm Michael, I love all things Science, Engineering, & 3D Printing. Staff Member. 0 and up, the software endstops are adjusted to preserve the physical movement limits. start gcode Slic3r single left. problem uploading. Working Subscribe Subscribed Unsubscribe 27. Billing and Coding Guidelines. A list of g-codes and m-codes for milling in the Fanuc, LinuxCNC, GRBL, and Haas dialects. G-Code Commands. However, any time you want to switch to a different extruder you must specify which. It is designed to be light to read (speed can be an issue) and versatile (it can control a nearly infinite amount of nozzles). -) A g-code editor built in so I could make small changes to the code before printing. This isn't the case, however, and in many cases you risk file corruption and data loss by doing this. Comments of this form must start and end on the same line: (Home some axes) G28 (here. gcode **** M73 P100 ; end build progress G1 Z155 F1000 ; send Z axis to bottom of machine M140 S0 T0 ; cool down HBP M104 S0 T0 ; cool down right extruder.


text:
## Introduction

The advance of high throughput sequencing has uncovered a large number of biomedically relevant DNA sequences, from driver mutations in cancer to new bacterial/viral pathogen DNA sequences to microbiome metagenomic profiles that affect mental disorders on the gut-brain axis1,2,3,4. For discovery applications, “shotgun” whole genome sequencing (WGS) is the preferred approach to identify novel DNA sequences of interest5. However, the human genome comprises over 3 billion nucleotides, and despite the lowering costs of high-throughput sequencing, it is not practical today to perform WGS to high depths necessary for identification of subclonal mutations, such as somatic mutations in cancer. For routine detection of disease-relevant DNA variants in known genes of interest, targeted sequencing or direct qPCR approaches are typically used6,7. Of the two dominant methods today for target enrichment, multiplex PCR tends to have shorter workflows and require less DNA input than hybrid-capture probes8. However, multiplex PCR struggles to scale to large panels covering hundreds of genes, due to the nonlinear increase of primer dimer species that reduce NGS mapping rates and increase effective cost9.

Currently, multiplex PCR methods for NGS target enrichment (e.g., Ampliseq8) primarily rely on (1) enzymatic digestion of modified bases in primers10 and (2) DNA size selection to preferentially remove short amplicon species likely to be primer dimers. However, both steps are labor-intensive and cannot be applied universally to all multiplexed PCR reactions. In contrast, relatively little systematic work has been reported on computational approaches to minimizing the formation of primer dimers in the first place. To the best of our knowledge, existing multiplex primer design algorithm never exceeded 70 primer pairs in one tube11,12,13,14. This is mainly due to the high computational cost when the number of primers increases15. The development of a robust multiplex primer set design algorithm that produces highly multiplexed primer sets with minimal primer dimer formation could allow further scaling of multiplex PCR target enrichment to even larger NGS panels when combined with enzymatic and size selection methods. Alternatively, it can simplify the workflow of moderate size NGS and qPCR assays by removing the need for strict contamination control from open-tube steps.

There are two primary challenges in designing highly multiplexed PCR primer sets: First, for an N-plex PCR primer set comprising 2N primers, there are $$\left(\begin{array}{l}2N\\ 2\end{array}\right)$$ possible simple primer dimer interactions. For N = 50, this corresponds to $$\left(\begin{array}{l}100\\ 2\end{array}\right)$$ = 4950 times as many potential primer dimer bindings as for a single-plex PCR primer set. Second, there are typically M > 10 reasonable candidate choices for each primer when considering specific gene targets and amplicon length constraints, resulting in M2N possible N-plex primer sets. For M = 20 and N = 50, the number of possible primer sets is 20100 ≈ 1.3 × 10130, billions of times larger than the number of atoms in the universe. Thus, it is computationally intractable to evaluate all possible multiplex primer sets. Simultaneously, primer dimer formation emerges from the interactions of two or more primers in the primer set, so changing the sequence of a primer to mitigate one primer dimer interaction may result in the appearance of another more serious primer dimer. In the language of numerical optimization, multiplex primer design is high dimensional problem with a highly non-convex fitness landscape. Consequently, standard convex optimization algorithms (e.g., gradient descent) will not be effective.

Here, we present Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), an algorithmic framework for designing highly multiplex PCR primer sets. Within this framework, we present an example multiplex primer design algorithm, comprising an algorithm for primer candidate generation and a rapidly computable Loss function for estimating primer dimers. Using the SADDLE, we designed and experimentally tested multiplex primer sets comprising 192 primers (96-plex) and 784 primers (384-plex), and show low primer dimer formation through NGS experiments. Building upon this success, we built a single-tube 60 primer qPCR and Sanger assay to detect and identify 56 gene fusions with clinical actionability for non-small cell lung cancer.

## Results

### Simulated Annealing Design using Dimer Loss Estimation (SADDLE)

There are six main steps in SADDLE, as illustrated in Fig. 1:

1. 1.

Generation of forward primer (fP) and reverse primer (rP) candidates for each gene target.

2. 2.

Selection of an initial primer set S0 from the primer candidates.

3. 3.

Evaluation of the Loss function L(S) on the initial primer set S0.

4. 4.

Generate a temporary primer set T based on set Sg (primer set from generation g) by randomly changing 1 or more primers.

5. 5.

Evaluate L(T), and set Sg+1 to either Sg (no change) or T, depending probabilistically on the relative values of L(Sg) and L(T).

6. 6.

Repeat steps 4 to 5 until an acceptable primer set Sfinal is constructed.

The above abstract framework provides a basis for many potential multiplex primer design algorithms, depending on the specific details of primer candidate generation, form of Loss function, temporary set T generation, and the dynamic probability of setting Sg+1 to T. Below, we describe our specific implementation of SADDLE, based on our accumulated understanding of primer design principles and primer dimer formation mechanisms. Given the infinite possibilities for function forms and hyper-parameters, we did not systematically evaluate or optimize at the high-level. Lower-level parameters, such as standard free energy (ΔG°) ranges for primers, were experimentally optimized and these are described below.

1. Primer candidate generation. We begin our implementation of primer candidate generation through the selection of one or more “pivot” nucleotides on human genomic DNA around which we design the forward and reverse primers (Fig. 2a). The pivot nucleotides are the ones that must be included in the amplicon insert, and for example could be the hotspot region of a gene that is frequently mutated. From the pivot nucleotides and a constraint on the maximum length of the amplicons (e.g., determined by the read length of NGS), we can systematically generate a series of different proto-primers with 3′ end just outside the pivot nucleotides. The proto-primers have a large range of different lengths and binding energies to their complementary sequences, and will next be trimmed at the 3′ end to generate the primer candidates (Fig. 2a).

From our past experiences and preliminary optimization experiments, primers that hybridize to their cognate templates with ΔG° ≈ −11.5 kcal/mol have the best tradeoff between amplification efficiency/uniformity and nonspecific hybridization. Shorter primers may not bind consistently with high efficiency to their templates, resulting in variability in amplification efficiency and non-uniformity of amplicon on-target reads in the NGS library. Longer primers have increased likelihood of binding to other loci in human genome, and can result in non-specific amplicons. Based on this ΔG° goal, we next systematically constructed primer candidates from the proto-primers by truncating nucleotides from the 3′ end until the primer candidate has ΔG° between −10.5 kcal/mol and −12.5 kcal/mol (Supplementary Section S9). Due to the granularity of ΔG° for base stacks, some proto-primers with the same 5′ end will result in multiple primer candidates (e.g., with ΔG° = −10.9 kcal/mol and −12.0 kcal/mol). Optionally, one could implement additional filters here to remove undesirable prime candidates, such as based on G/C content. For our demonstration panels, we restrict the G/C content of primer candidates to be between 0.25 and 0.75, removing primer candidates with G/C content outside this range.

In the implementation of SADDLE, primer candidates can be treated as individual primers or as primer pairs. Our specific implementation treats primers as pairs, so we next combinatorially generate all candidate primer pairs for an DNA target, in order to better constrain the distribution of amplicon lengths. Any candidate primer pairs that generate amplicons with length exceeding our maximum amplicon length or below our minimum are removed.

2. Initial primer set S0 selection. We randomly selected a primer pair candidate for every amplicon that we wish to design, and collectively the selected primers are known as the initial primer set S0.

3. Evaluation of Loss function L(S) on S0. The Loss function L(S) is a rapidly computable function that aims to approximate the severity of primer dimer formation by a primer set S. L(S) sums the potential primer dimer interactions between every pair of primers in the primer set. To prevent confusion, we refer to the predicted formation of dimers for a particular pair of primers to be the Badness. Mathematically,

$${{\mbox{L}}}\,(S) =\mathop{\sum}\limits_{b\ge a}\,{{\mbox{Badness}}}\,({p}_{{{{{{\mathrm{a}}}}}}},{p}_{{{{{{\mathrm{b}}}}}}})\\ =\frac{1}{2}\cdot \mathop{\sum }\limits_{a=1}^{2N}\mathop{\sum }\limits_{b=1}^{2N}\,{{\mbox{Badness}}}\,({p}_{{{{{{\mathrm{a}}}}}}},{p}_{{{{{{\mathrm{b}}}}}}})+\underbrace{\frac{1}{2}\cdot \mathop{\sum }\limits_{a = 1}^{2N}{{\mbox{Badness}}({p}_{{{{{{\mathrm{a}}}}}}},{p}_{{{{{{\mathrm{a}}}}}}})}}_{\begin{array}{c}{{\mbox{pre-calculated}}}\end{array}}$$
(1)

where pa and pb are the ath and bth primer in primer set S, respectively (Fig. 1). Note that the second term of L(S) can be calculated in advance during primer candidates generation. One can imagine the Badness function to be proportional to the amount of primer dimers formed by two primers. In an optimized primer set with a relatively low concentration of primer dimers compared to the concentration of on-target amplicons, the amount of primer dimers formed between primer pa and pb should not significantly impact the amount of dimers formed between pa and pc, so the Loss function being defined as the sum of the component Badness functions is justified.

The Badness function in our implementation is defined as follows (Fig. 2b):

$$\,{{\mbox{Badness}}}({p}_{{{{{{\mathrm{a}}}}}}},{p}_{{{{{{\mathrm{b}}}}}}})=\sum \frac{{2}^{{{{{\mathrm{len}}}}}}\cdot {2}^{{{{{\mathrm{numGC}}}}}}}{({d}_{1}+1)({d}_{2}+1)}$$
(2)

The sum in the Badness definition is over all reverse complementary subsequences between primer pa and pb with at least 4 nt of continuous complementarity. len is the length of the subsequence, d1 and d2 are the distances of the subsequence to the 3′ ends of primer pa and pb, respectively, and numGC is the number of G/C nucleotides in the subsequence. Our choice of 4 nt is based on our preliminary experimental studies in qPCR showing that up to 3 nt of complementarity at the 3′ ends of two primers will not result in significant primer dimers even in no-template control (NTC) reactions.

For each complementary subsequence, its length (len) and its number of GC nucleotides in complementary subsequence (numGC) contribute exponentially to Badness. Thus, the exponential components of Badness roughly reflect the partition function of the complementarity interaction, with G/C base pairs roughly twice as strong as A/T base pairs. We chose to use these simplistic parameters, rather literature base stacking thermodynamics parameters16,17,18, because there is significant uncertainty in the effective salinity of the PCR reaction buffer, and because our previous studies on DNA thermodynamics suggests that previously reported ΔH° and ΔS° parameters do not extrapolate well to higher temperatures19.

The distances of the complementary subsequence to the 3′ ends of primers pa and pb, denoted as d1 and d2, are known to significantly affect the likelihood of primer dimer formation. In our preliminary qPCR experiments, we observed that a primer pair with 10 nt of complementarity at the 5′ end will not result in observable primer dimer formation, but a primer pair with 5 nt of complementarity at the 3′ end would. Depending on whether the specific DNA polymerase used, different d1-based and d2-based attenuation of Badness may be optimal for minimizing primer dimers. Because high-fidelity DNA polymerases with $${3}^{\prime} > \, {5}^{\prime}$$ exonuclease activity can remove mismatched 3′ nucleotides, the optimal d1-based and d2-based attenuation should be significantly weaker for high-fidelity DNA polymerases.

The evaluation of the Badness function is single largest component of software runtime cost, due to the large number of times the Badness function will be evaluated. For a primer that is 25 nt in length, there are 22 subsequences of length 4, 21 subsequences of length 5, etc. Evaluation of Badness for a single primer pair would thus have time complexity of O(P3), where P is the length of each primer (Eq. 2). In our specific implementation, subsequence length len also has a maximum of 8 nt, decreasing the time complexity to O(P2). Naively, evaluation of the Loss L(S) of the whole primer set would have time complexity of O(N2 × P2) (Eq. 1). However, due to the additive nature of subsequence components to the overall Badness function, we implement more rapid Badness evaluation by using a hash table20, as shown in Eq. (3), where H is the hash table, s is a subsequence of the primer set, d is the distance to 3$${}^{\prime}$$ ends of each occurrence of s, and revcomp is a function that converts s to its reverse complement sequence (Fig. 2b). The time complexity to set up the hash table is O(N × P) to calculate the hash value for each unique subsequence in the primer set, and the time complexity to evaluate the L(S) by stepping through all subsequences of all primers is also O(N × P) (Eq. 3). Consequently, the overall time complexity of evaluating L(S) is O(N × P) for all primers in S.

$${{\mbox{H}}}\,[s]=\sum \frac{1}{d+1}\\ \mathop{\sum }\limits_{a=1}^{2N}\mathop{\sum }\limits_{b=1}^{2N}\,{{\mbox{Badness}}}({p}_{{{{{{\mathrm{a}}}}}}},{p}_{{{{{{\mathrm{b}}}}}}})=\sum \frac{{2}^{{{{{\mathrm{len}}}}}}\cdot {2}^{{{{{\mathrm{numGC}}}}}}}{d+1}{{\mbox{H}}}\,[\,{{\mbox{revcomp}}}\,(s)]$$
(3)

4. Generate temporary primer set T based on Sg. Step 4 begins the recursive optimization process. Based on the current primer set Sg at generation g, we first randomly select one primer pair to “mutate.” For that primer pair, we randomly select a different primer pair from the list of all candidate primer pairs generated in Step 1. Temporary primer set T is thus generated by combining this new primer pair with all remaining primers in set Sg. Optionally, multiple primer pairs can be replaced simultaneously in this step to allow faster and more efficient exploration of the primer set space. In our preliminary in silico evaluations, we found that simultaneously mutating multiple primer pairs generally caused a slowdown of the optimization process.

5. Evaluate L(T) and set Sg+1 to be either T or Sg. The Loss of temporary primer set T can be evaluated significantly faster than the initial evaluation of L(S0), because the hash table only need to be modified based on the changed primers.

We next compare the value of L(T) vs. L(Sg). If L(T) is smaller than L(Sg), then the primer pair change was an improvement and accepted, so Sg+1 is set to T. If L(T) is larger than L(Sg), the change was detrimental, but we will still accept the change with a certain probability, as part of the simulated annealing algorithm21. To clarify, “simulated annealing” here refers to a specific computer science algorithm, and not a literal simulation of a physical DNA thermal annealing process. If we never accept any detrimental primer pair changes, then the approach degenerates to become a stochastic gradient descent approach. In preliminary in silico evaluations, we confirmed that stochastic gradient descent produces final primer sets with significantly worse Loss, because it becomes too easy to get stuck in a local Loss minima.

The probability of accepting a detrimental change depends on both the magnitude of the detriment (L(T) − L(Sg)) and the generation g of the optimization. Worse changes with higher L(T) are accepted with lower probability, and later generations of the optimization (higher g) are less tolerant of detrimental changes. In our implementations, the probability of setting Sg+1 to be T when L(T) is greater than L(Sg) are as follows:

$$p=\left\{\begin{array}{ll}{e}^{\frac{{{{{{{\rm{L}}}}}}}({S}_{{{{{{\mathrm{g}}}}}}})-{{{{{{\rm{L}}}}}}}(T)}{{{{{{{\rm{C}}}}}}}(g)}}&\,(g \; < \; {g}_{{{{{{\mathrm{t}}}}}}})\\ 0&\,(g\;\ge\; {g}_{{{{{{\mathrm{t}}}}}}})\end{array}\right.$$
(4)

where e is Euler’s number and gt is a positive interger. C(g) is a function that is monotonically non-increasing in g, indicating decreasing tolerance to detrimental changes at later generations. The parameter gt indicates the generation in which simulated annealing terminates, and we switch over to stochastic gradient descent.

6. Repeat steps 4 and 5. Steps 4 and 5 are repeated until either a pre-determined generation g, or until L(Sg) is below a pre-determined threshold Lt. In our implementation, we typically run the optimization to about 1.5 × gt to ensure we reach local minima. To further improve the overall quality of the generated primer set, we recommend running multiple SADDLE optimization processes with different starting conditions (initial primer sets) and selecting the best final primer set.

### Design and experimental evaluation of a 96-plex primer set

We first used SADDLE to optimize the design of a 96-plex primer set, each amplicon target one arbitrarily selected exon of a different cancer-related gene22,23,24,25 (Fig. 2c). Figure 2d shows the calculated value of L(Sg) at different generations g, and is representative of our typical optimization trajectory. We selected the designed primer sets at three different optimization generations for experimental testing: PS1 (initial unoptimized primer set), PS2 (primer set with intermediate Loss optimization), and PS3 (primer set with saturating Loss optimization). The primer set Loss decreased roughly 24-fold from PS1 to PS3; after 40,000 generations, only very marginal improvements were observed. We chose the primer set at 40,000 generations as PS3, rather than the one at 60,000 generations, because we know that our Loss function is an imperfect predictor of primer dimers. Over-training on an imperfect Loss function can lead to worse experimental results. The optimization finished in about 10 min under a conventional laptop with MATLAB R2021b and Linux operating system.

We applied each of the three primer sets individually to human genomic DNA (10 ng NA18562, sheared to a mean length of approximately 150 nt) and amplified for 17 cycles. We next constructed NGS libraries from the amplicons generated using PS1, PS2, and PS3, using a standard adaptor ligation protocol (Supplementary Section S1). After library preparation, capillary electrophoresis results show a clear increase of amplicons of the expected length from PS1 to PS2 to PS3 (Fig. 2e). In the NGS data analysis workflow, after the first step of adapter trimming, we separated NGS reads into three major species: on-target amplicons, dimers, and non-specific amplicons (Supplementary Section S2). On-target amplicons are the NGS reads that were successfully aligned to the intended amplicon sequences using Bowtie226. The remaining NGS reads were aligned separately to each forward and reverse primer sequence. Reads with insert length shorter than the sum of the two aligned primers are classified as Dimers, and reads with insert length longer than the sum of the two aligned primers are classified as Non-specific amplicons (amplifying unintended regions of the human genome).

The amounts of these three species in the three primer set libraries are shown in Fig. 3a. Going from the PS1 to the PS3 library, the fraction of primer dimers dropped significantly, from 90.7% in the PS1 library to 39.6% in the PS2 library and then to 4.9% in the PS3 library. However, even with the decrease of dimers from the PS2 library to the PS3 library, the proportion of non-specific amplicons in these two libraries remained about the same. This is reasonable because the SADDLE Loss function was designed only minimizes primer Dimers, and does not consider likelihood of Non-specific amplicon formation. The distribution of amplicon length in NGS reads is consistent with the capillary electrophoresis results in three libraries (Fig. 3b and Supplementary section S3).

We next tested the PS3 primer set on five formalin-fixed, paraffin-embedded (FFPE) clinical tissue samples (one breast cancer, two lung cancer, and two colorectal cancer samples, see also Supplementary Section S5). The beeswarm plot of the observed reads (Fig. 3d) show high consistency across the different samples, and are also consistent with our results from sheared genomic DNA. The identities and quantities of primer dimers formed, likewise, are similar between FFPE DNA samples and genomic DNA (Fig. 3e).

To demonstrate the scalability of SADDLE, we next designed and tested a 384 amplicon panel comprising 768 primers. The optimization finished in about 60 min under a conventional laptop with MATLAB R2021b and Linux operating system. Due to the high cost of primer synthesis for this large panel, we only experimentally tested the final primer set design. Surprisingly, the observed Dimer fraction was only 1% for this library, using an input of 40 ng sheared NA18562 genomic DNA (Fig. 3f). Roughly 56% of the reads were Non-specific amplicons, resulting in a NGS library on-target rate of 43% (Supplementary section S6).

### Accuracy of the dimerization prediction

We constructed the SADDLE Badness function based on our understanding of the mechanisms of primer dimer formation, but we know that this Badness function is imperfect both because our understanding of primer dimer formation is imperfect, and because it is computationally too expensive to implement many classes of potentially more accurate Badness functions. Accurate assessment of how good or bad the current Badness function is at predicting Dimers, however, is critical to further incremental improvement in multiplex PCR primer design using SADDLE.

Through the course of SADDLE optimization, we expect that the Dimer prediction accuracy will get worse in later optimization generations, because we are selecting for primer sets with low expected Badness that will include false negatives. Experiments and analysis of PS1, PS2, and PS3 confirm this understanding (Supplementary Section S7). The Dimer reads for each pair of primers from PS1 are plotted against the predicted Badness in Fig. 4a.

To facilitate discussions of Badness function accuracy in terms of sensitivity and specificity, we set two separate thresholds: the Reads Threshold (horizontal orange line) and the Badness Threshold (vertical dotted purple line). The plotted Reads Threshold in Fig. 4a corresponds to the mean on-target read depth, and the Badness Threshold plotted correspond to the value that maximizes prediction sensitivity plus specificity. For these Threshold values, we observe a sensitivity 92.5% ($$\frac{62}{67}$$) and a specificity of 90.3% ($$\frac{33,226}{36,797}$$). By adjusting the Badness Threshold value, we can change the tradeoff between sensitivity and specificity, resulting in a receiver operator characteristic (ROC) curve (Fig. 4b). The area under the ROC curve (AUROC) is 0.9577, indicating very high Dimer prediction accuracy by the Badness function. When the Read Threshold is adjusted higher, the AUROC also increases (Fig. 4c), but the positive predictive value (PPV) decreases.

We next examined the top five most dominant Dimer reads in the library (Fig. 4d) and compared them to the top five predicted dimer reads based on the Badness function (Fig. 4e). It is noteworthy that only one of the two different top five lists overlap. The other four predicted dimers did not contribute significantly experimentally, and the other four observed dimers were not predicted to have high risk for dimer formation. At a glance, it appears we over-weighted the possibility of forming primer dimers in which the 3′-most nucleotide in unpaired, and we may need to adjust the Badness function to allow a stronger attenuation of Badness based on distance from the 3′ end. Additionally, it appears that the Badness function may be not scaled optimally, as the log10(Badness) ranges between 0 and 3.5, whereas the log10(Dimers) ranges between 0 and 5 (Fig. 4f). This may mean that the current algorithm over-weights weak potential dimers, at the expense of insufficiently avoiding strong predicted primer dimers.

Beyond the above observations, it is not clear why some dimers are observed at much higher reads experimentally than others. For example, the top observed dimer only has a 5 nt overlap at the 3′ end, compared to a 7 nt overlap at the 3′ end for the rank 4 dimer. This is not consistent with our understanding of DNA hybridization and polymerase extension kinetics, and implies that we may not be able to generate a perfect Badness function even ignoring computational resource constraints.

### Gene fusion detection with qPCR and sanger sequencing

Gene fusions are therapeutic targets and attractive diagnostic biomarkers to guide treatment27,28,29,30. Currently, gene fusions are detected either in single-plex by qPCR for known high-frequency fusions (e.g., BCR-ABL1), or by NGS. A highly multiplexed qPCR assay that can detect tens of potential gene fusions relevant to a particular disease could greatly increase the accessibility of gene fusion testing.

Here, we used SADDLE to design a set of 60 primers to detect 56 actionable gene fusions for non-small cell lung cancer (NSCLC) across six genes (ALK, ROS1, RET, NRTK1, NTRK2, and NRTK3). The number of primers are lower than 56 × 2 because the same exon can be fused with multiple partner genes or exons. We detect the fusions in complementary DNA (cDNA) reverse transcribed from RNA, in order to limit the complexity and length of the detection targets. For each fusion of interest, the primer set includes a forward primer (fP) targets the upstream partner gene and a reverse primer (rP) targets the downstream partner gene (Fig. 5a).

We first tested the multiplex PCR panel against synthetic samples bearing the gene fusions of interest (Fig. 5b, c). In all cases, the positive samples were clearly distinguishable by cycle threshold (Ct) value against both commercial wildtype cDNA (WT) and the no-template control (NTC), with all ΔCt values above 10. We also tested the panel on synthetic gene fusion samples with a variant allele frequency (VAF) of 1% (Supplementary Section S8). The 1% VAF samples were constructed by mixing synthetic gBlocks that contained a single fusion (the variant) with human cDNA (the wildtype).

Finally, we applied the gene fusion qPCR panel to clinical cDNA samples extracted from extracellular vesicles in blood plasma from NSCLC patients (Fig. 5d). Of the ten clinical samples analyzed, three were called positive for gene fusions. To identify the exact gene fusion in these samples, we performed Sanger sequencing on the amplicons from the positive samples. Two samples were identified with EML4 exon20-ALK exon20, and one was identified with EML4 exon 15-ALK exon 20.

## Discussion

In this study, we designed a multiplex PCR primer design algorithm SADDLE targeting numerous genomic regions in a single tube. We presented experimental validation of primer sets on a 96-plex cancer-related exons panel, demonstrating that the SADDLE was capable in selecting better primers by reducing dimerization in a multiplex PCR reaction. The dimer rate decreased going from the 90.7% in a naively designed PS1 to 39.6% in an intermediate PS2 and to 4.9% in an optimized PS3, resulting in an increased on-target rate as well as greater uniformity of on-target amplicons. In another 384-plex panel targeting random-selected SNPs in the human genome, the NGS library using the optimized primer set showed a dimer rate of 1%. SADDLE can reduce reagent costs and enable the amplification of hundreds of target templates simultaneously without wasting NGS reads. Importantly, library preparation using optimized primer sets generated by SADDLE does not depend on labor-intensive enzymatic cleavage or size selection steps to remove dimers.

The improvement of NGS library on-target rates through the reduction of primer dimers can allow significantly larger targeted panels to be possible using multiplex PCR library preparation. Because multiplex PCR generally requires less input DNA and are faster than ligation-based library preparation approaches31, due to the low yields of end repair and ligation, we envision that SADDLE-designed primers can be useful for a variety of research and clinical applications where DNA sample quantities are limited and/or where rapid turnaround is needed. For example, in oncology tissue biopsies obtained through fine needle aspirates and core biopsies are frequently insufficient for standard NGS analysis, and cell-free DNA from peripheral blood plasma likewise are limited and impose sensitivity limitations to ligation-based approaches32. Furthermore, in reproductive medicine, samples from amniocentesis and preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD) are also very limited, and require rapid turnaround for molecular diagnostics due to the time-sensitivity of clinical decisions33.

Through our analyses of predicted vs. observed dimers, we found that the parameters in the Loss function used in SADDLE could be adjusted to optimize dimer prediction performance, particularly in the 3$${}^{\prime}$$ distance attenuation. However, with the current SADDLE algorithm, Non-specific amplicons now appear to dominate off-target rates, rather than Dimers. Thus, to further scale-up the panels that can be designed by SADDLE, it will be necessary to construct and optimize new Loss functions that penalize primer sets based on predicted off-target genomic amplification. Modification of the Loss function to minimize Non-specific amplicon formation would require significantly more work, as it requires consideration of the expected sample genome sequence. Whereas the current Loss function is “universal” in improving multiplex PCR primer set designs, a Loss function that considers Non-specific amplicons would inherently be suboptimal for primer dimer minimization. A Loss function predicting Non-specific amplification must also consider external factors, including the average length of the DNA molecules in the sample and nonpathogenic genomic polymorphisms. Current Loss function can be further improved based on NGS data and other methods including machine learning34.

In medical and research applications where the cost of NGS cannot be economically justified35, qPCR assays will likely be the dominant tool for study of genomic variants. In qPCR, even single-plex primers can form significant dimers if poorly designed with Ct values below 30. Multiplex qPCR thus typically requires significant empirical optimization, even at around 4-plex11. SADDLE allowed us to successfully design a 60-primer qPCR panel targeting 56 gene fusions, and exact fusion identities can be determined through affordable Sanger sequencing. Thus, we envision that SADDLE can revolutionize the use of qPCR for highly multiplex molecular diagnostics.

## Methods

### Ethical approval

All procedures performed in studies involving human participants were approved by the ethics committees of Shanghai Pulmonary Hospital, Tongji University (protocol K19-155Y), and were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all participants.

### Oligonucleotides

All primers were purchased as standard desalted DNA oligonucleotides (Integrated DNA Technologies), and stored at 4 °C.

### Samples

Synthetic DNA templates were purchased as desalted DNA oligonucleotides (gBlocks, Integrated DNA Technologies), and stored at −20 °C. Human cell-line gDNA sample NA18562 (Coriell Biorepository) was stored at −20 °C. The gDNA was mixed with synthetic DNA templates at various ratios to create samples containing different proportions of a specific variant sequence. Dilution of gDNA samples and synthetic DNA templates were made in 1× TE buffer with 0.1% Tween 20 (Sigma Aldrich).

FFPE slides were purchased from Coriell Institute. FFPE DNA was extracted from GeneRead DNA FFPE Kit (Qiagen).

Ten plasma samples from ten NSCLC patients in de-identified format were collected from Shanghai Pulmonary Hospital. RNA in extracellular vesicles was extracted with exoRNeasy Serum/Plasma Kit (Qiagen). cDNA was synthesized with SuperScript IV First-Strand Synthesis System (ThermoFisher Scientific).

### Multiplex PCR protocol

Multiplex PCR was performed on a T100 Thermocycler or a C1000 Thermocycler (Bio-Rad). The total volume of each reaction was 50 µl. DNA sample input ranged from 10 to 100 ng per tube. PCR reagents including vent (exo-) polymerase, ThermoPol Reaction Buffer (10×), and dNTP (New England Biolabs) were used for enzymatic amplification. Thermal cycling started with a 3 min incubation step at 95 °C for polymerase activation, followed by 17 cycles of 30 s at 95 °C for DNA denaturing, 3 min at 60 °C for annealing, and 30 s at 72 °C for extension, followed by a final extension of 5 min at 72 °C. Detailed experiment protocol for the NGS library preparation can be found in Supplementary Section S1, S3.

### End repair protocol

Multiplex PCR product was end-repaired using NEBNext® Ultra II End Repair/dA-Tailing Module (New England Biolabs). Each reaction was a mixture of 3 μl NEBNext Ultra II End Prep Enzyme Mix, 7 μl NEBNext Ultra II End Prep Reaction Buffer, 20 μl multiplex PCR products, and 30 μl H2O. End repair was performed on a Eppendorf Mastercycler. Thermal cycling started with the incubation at 20 °C for 30 min and 65 °C for 30 min, with the heated lid set to 80 °C.

End repair mixture was ligated with adapters using NEBNext® Ultra II Ligation Module (New England Biolabs). Each reaction was a mixture of 30 μl NEBNext Ultra II Ligation Master Mix, 1 μl NEBNext Ligation Enhancer, 2.5 μl NEBNext Adaptor for Illumina, and 60 μl previous End repair mixture. Ligation was performed on a Mastercycler from Eppendorf. Thermocycling started with the incubation at 20 °C for 15 min with the heated lid off; after adding 3 μl USER enzyme to the ligation mixture, the reaction was incubated at 37 °C for 15 min with the heated lid set to 55 °C.

### Index quantitative PCR

Following adapter ligation, Index qPCR was performed on CFX96 Touch Deep Well Real-Time PCR Detection system (Bio-Rad). Quantification of different libraries was performed simultaneously in each well. Each reaction was a 10 μl mixture, with 1 μl i5 index, 1 μl i7 index, 1 μl ligation products, 2 μl Milli-Q, and 5 μl PowerUp SYBR Green Master Mix. Experiment was performed following a thermal cycling protocol with a 3 min incubation step at 95 °C for polymerase activation, followed by 40 cycles of 10 s at 95 °C for DNA denaturing and 30 s at 60 °C for annealing and extension. Ct values were obtained directly from the CFX96 system.

### Index PCR

Index PCR was performed on a T100 Thermocycler or a C1000 Thermocycler (Bio-Rad). Index primers used were NEBNext® Multiplex Oligos for Illumina® (New England Biolabs). Each reaction was a mixture of 2 μl each i5 and i7 index primers, 5 μl ligation products, and PCR reagents including vent (exo-) polymerase, ThermoPol Reaction Buffer (10×), and dNTP. The volume of each reaction was 52 μl. Thermal cycling started with a 3 min incubation step at 95 °C for polymerase activation, followed by various cycles of 30 s at 95 °C for DNA denaturing and 30 s at 60 °C for annealing, and 30 s at 72 °C for extension, followed by a final extension of 5 min at 72°.

### Column purification

Multiplex PCR products and ligation products were all purified using DNA Clean & Concentrator Kits (ZYMO Research). The volume of DNA-binding buffer was 250 μl for multiplex PCR products clean-up, and 482.5 μl for ligation products clean-up; 25 μl Milli-Q water was used as elution buffer for each reaction.

Index PCR product was purified using AMPure XP beads (Beckman Coulter). For each 50 μl reaction mixture, 90 μl of beads was added; 40 μl Milli-Q water was used as elution buffer.

### Library quantitation

All the libraries were quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific).

### Bioanalyzer

Sizes of PCR products and libraries were measured using Bioanalyzer High Sensitivity DNA Assay (Agilent), and DNA chips were run on the Agilent 2100 Bioanalyzer system.

### Next-generation sequencing

All the libraries were loaded on a Miseq Reagent V2 for obtaining pair-end reads and were sequenced on a Miseq (Illumina).

### Sanger sequencing

PCR products were purified and prepared using a BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and were sequenced on a Thermo Fisher Scientific 3500 Series Genetic Analyzer. Detailed experiment protocol for Sanger Sequencing can be found in Supplementary Section S8.

### Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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text: I can only speak of my experience. I was first taught convergence of sequences and then later on, the definition of limits with $\varepsilon-\delta$ and its equivalent form involving sequences. I still find this most intuitive, but then this opinion is clearly heavily influenced by my upbringing.


text:
# Math Help - Slant Asymptotes

1. ## Slant Asymptotes

Show that the curve y = square root (x^2 +4x) has two slant asymptotes y = x + 2 and y = x - 2.

I am confused on where to start on this question, any help would be greatly appreciated, thanks so much in advance.

2. Originally Posted by Amybee
Show that the curve y = square root (x^2 +4x) has two slant asymptotes y = x + 2 and y = x - 2.

I am confused on where to start on this question, any help would be greatly appreciated, thanks so much in advance.
1. The second asymptote should read: y = -x - 2

2. The slope of the slant asymptote is calculated by:

$m = \lim_{ x \to \infty} \left(\dfrac yx \right) = \lim_{ x \to \infty} \left(\dfrac {\sqrt{x^2+4x}}x \right) = \lim_{ x \to \infty} \left(\dfrac {|x| \sqrt{1+ \frac4x}}x \right) = 1$
Or m = -1 which depends on the sign of x.

Cab you take it from here?

3. I think so.

4. Originally Posted by Amybee
I think so.
$y=\sqrt{x^2+4x} = \sqrt{(x^2+4x+4)-4}=\sqrt{(x+2)^2-4}$
$a = \sqrt{(x+2)^2}=|x+2|$


text:
Question

# The first ionization energy of $Na,B,N,$ and $O$atoms follow the order:A. $B < Na < O < N$B. $Na < B < O < N$C. $Na < O < B < N$D. $O < Na < N < B$

First ionisation energy of all the elements (like $(Na,{\text{B,N}}$ and ${\text{O)}}$ depends upon electronic configuration.First of all, $Na$is an electropositive element, or alkali metal and belongs to the first group of the periodic table. Size of $Na$is larger than that of other elements and lesser force of attraction between the nucleus and outermost electrons. So its ionisation energy becomes less than that of other elements. But as we move from left to right in a period, nuclear charge increases and atomic size decreases thus ionisation energy increases.
Along the period, ionisation energy increases then the order of elements i.e. $Na < B < N < O$ but the actual order of $I.{E_s}$ is $Na < B < O < N$, the reason being half filled configuration $\left( {2{s^2}2{p^3}} \right)$of nitrogen followed by oxygen due to its smaller size than boron.
Note:: The force of attraction between the nucleus and outermost electron increases with increase in nuclear charge. Greater is the nuclear charge, greater will be the energy required to pull the electron from the atom. Hence, with increase in nuclear charge ionization enthalpy increases. However, the effective nuclear charge experienced by a valence electrons in an atom will be less than the actual charge on the nucleus due to a phenomenon known as shielding effect’ or screening effect’ where the force of attraction by the nucleus on the valence electron is reduced due to the repulsive forces exerted by the inner shell electrons.


text:
#### [SOLVED] Lagrangian formalism (demonstration)

My question is about the multiplicity of the Lagrangian to a Physics system.

I pretend to demonstrate the following proposition:

For a system with $n$ degrees of freedom, written by the Lagrangian $L$, we have that: $$L'=L+\frac{d F(q_1,...,q_n,t)}{d t}$$ also satisfies Lagrange's equations, where $F$ is any arbitrary function, but differentiable.

I saw the resolution to this problem and I found that another interesting proposition was needed to complete the demonstration, and it was:

$$\frac{\partial \dot{F}}{\partial \dot{q}}=\frac{\partial F}{\partial q}$$

Why is this true? How could I say that the relation between the temporal variation of $F$ and the temporal variation of $q$, is equivalent to the relation between $F$ and $q$.

Maybe this is a silly question, but I don't get it in my head. I can try examples and see that it's true, but I can't figure it out why.

#### @user12029 2015-04-06 00:20:07

Hint: $\frac{\partial }{\partial \dot{q}}\dot{F}=\frac{\partial}{\partial \dot{q}}\left(\frac{\partial F}{\partial q}\dot{q}+\frac{\partial F}{\partial \dot{q}}\ddot{q}+\frac{\partial F}{\partial t}\right)$

What does $F=F(q,t)$ imply about $\frac{\partial}{\partial \dot{q}} F$?


text:
# Order and generators of intersection of cyclic groups

Let $\sigma$, $\tau$ be two permutations of $S_n$.

We know that $\langle \sigma \rangle \cap \langle \tau \rangle$ is a cyclic subgroup and (from Lagrange's Theorem) that its order divides $$\gcd(\left|\langle \sigma \rangle\right|, \left|\langle \tau \rangle\right|).$$

Do we have other results that offer us a way to find $k$, $h$ such that $$\langle \alpha\rangle = \langle \sigma \rangle \cap \langle \tau \rangle = \langle \sigma^k \rangle = \langle \tau^h \rangle,$$ that is, to find the order and a generator of the intersection?

For example: how would we proceed if we consider in $S_{15}$ $$\sigma = (1,2,3,4)(5,6,7,8,9)(10,11,12)(13,14)$$ $$\tau = (1,3) (2,4)(5,6,7,8,9)(10,12,11)(13,14,15)$$ and want to find $\langle \sigma \rangle \cap \langle \tau \rangle$? From the Lagrange's theorem we only know that its order divides $30$.

When proving that the intersection of two cyclic subgroups is again cyclic, one can do it constructively by showing that (I'm just using your notation above)

$$\sigma^k, k :=\min\{m \in \mathbb{N}_{\geq 1} \mid \sigma^m \in \langle \alpha \rangle\}$$

is a generator of $\langle \alpha \rangle$.

So regarding your example, we are looking for a minimal $m$, such that $\sigma^m = \tau^n$ for an $n \in \mathbb{Z}$. Since we are dealing with cycles, we can assume that $n \in \mathbb{N}$. Now $\sigma^m(15) = 15$ $\forall m$, so looking at the last cycle of $\tau$, which is $(13, 14, 15)$, $n$ must be a multiple of 3. Then $\tau^n(j) = j$ $\forall j \in \{10,...,15\}$. In order to achieve this for $\sigma^m$ as well, $m$ must be a multiple of 2 and 3, so $6\mid m$. Assume $m = 6$. This leaves the 5-cycle $(5,6,7,8,9)$ unaltered, so $n \equiv 1 \bmod 5$.

In fact

$$\sigma^{6} = (1,3)(2,4)(5,6,7,8,9) = \tau^{21}.$$

Now $\langle \alpha \rangle = \langle \sigma^6 \rangle$ with $\mathrm{ord}(\sigma^6) = 10$.

• Hi, I'm trying to solve a similar problem and I'm trying to apply the reasoning you explained. The only thing that I haven't clear is the point where you say $\sigma^m(15) = 15$. Why do you affirm this? Because $\sigma$ fixes 15 or because of the two permutations belonging to $S_{15}$? Thanks. – PCNF Jan 26 '19 at 15:54
• @PCNF A bit old, sorry, but still: Yes, because $\sigma$ fixes $15$. – johnnycrab Feb 20 '19 at 8:20


text:
# Composite function of a piecewise function

1. Jan 16, 2014

### libragirl79

1. The problem statement, all variables and given/known data

Given that I have a doubling function :
f(x)=2x (for 0≤x<0.5) and 2x-1 (for 0.5≤ x<1)

2. Relevant equations
What is f(f(x))?

3. The attempt at a solution
f(f(x))=4x for the first one and 4x-3 for the second part but not sure what to do about the domain constraints...

Thanks!!

2. Jan 16, 2014

### SammyS

Staff Emeritus
It's not that simple.

Yes, f(f(x)) = 4x over part of the domain of the composite function and 4x-3 over some other portion, but those are not the only two pieces of f(f(x)).

For what values of x is 0 ≤ 2x < 0.5 ?

For what values of x is 0.5 ≤ 2x < 1 ?

etc.

3. Jan 17, 2014

### libragirl79

right, that's exactly my issue, since i don't have the fcns for 1/4 ≤ x < 1/2
and 1/2 ≤ x < 3/4 ... is there a certain method for doing this?

4. Jan 17, 2014

### HallsofIvy

Staff Emeritus
You understand that "$1/4\le x< 1/2$" is part of the interval $0\le x< 1/2$ don't you?

If $0\le x< 1/4$, f(x)= 2x which is less than 1/2 so ff(x)= f(2x)= 2(2x)= 4x.
If $1/4\le x< 1/2$ then x is still between 0 and 1/2 so f(x)= 2x but f(x) is now between 1/2 and 1 so ff(x)= f(2x)= 2(2x)- 1= 4x- 1.

Do similarly for $1/2\le x< 3/4$. Now x is between 1/2 and 1 so f(x)= 2x- 1 which is between 0 and 1/2.

If x is between 3/4 and 1, f(x)= 2x- 1 is between 1/2 and 1

5. Jan 18, 2014

### libragirl79

I understand the breakdown of the x domains, but how do you know where f(x) falls?