If your USB drive size is less than 137GB, then just use the \MAKE_E2B_USB_DRIVE (run as admin).cmd script. See here.
If your USB drive is over 137GB (Flash drive or HDD), then use RMPrepUSB to create a 130GB NTFS partition (do NOT tick boot as HDD C: option) and then use another tool to create a second Primary partition. See here. This is because many BIOSes have a 137GB limit for USB drives (even recent BIOSes!).
Unless you have specific requirements (such as you boot to DOS\FreeDOS a lot), I recommend you choose NTFS for your E2B USB drive and not FAT32.
You will still be able to UEFI-boot from .imgPTN files if your USB drive is formatted as NTFS.
I do not recommend exFAT for E2B (though it should work) because some Operating Systems cannot access exFAT partitions.
4. Adding Payloads
Always check the list here for your particular payload and any special instructions.
Always run \MAKE_THIS_DRIVE_CONTIGUOUS.cmd before booting.
If you want to UEFI-boot and MBR-boot, make a FAT32 .imgPTN file using the MPI Tool Kit.
UEFI-booting will only work if the payload supports it and contains UEFI-boot files.
Booting a file as .ISO may not work correctly if you have a Fixed disk type of E2B USB drive.
.imgPTN files will work on Removable or Fixed disks.
Some payloads do not support UEFI-booting, but if you use the grub2 menu system, it is possible to UEFI-boot to many more linux ISOs.
Below are listed some typical payload files - it is important to know what type of USB drive you have first (Removable or Fixed) because this can affect how they boot...
All types of E2B drive (Removable and Fixed types):
Linux ISOs (MBR booting) - Copy the ISO to any of the standard menu folders (e.g. \_ISO\MAINMENU or \_ISO\LINUX).
Linux ISOs (UEFI-booting) - Copy the .imgPTN file to any of the standard menu folders (e.g. \_ISO\MAINMENU or \_ISO\LINUX).
Puppy Linux ISOs - use the .isopup file extension
Mac puppy - use .isomacpup file extension
Linux + persistence - Find and read the nearest matching .mnu file in the \_ISO\docs\Sample mnu Files\Linux folder.
Windows XP 32-bit Install ISOs (MBR-booting) - Copy to \_ISO\WINDOWS\XP folder
Windows To Go VHD - Copy the .VHD file to any of the standard menu folders (e.g. \_ISO\MAINMENU or \_ISO\WIN).
ERD Commander 2005/2007/MSDaRT5 - use the .isomemF01 or .isomemwinv file extension or use the ERD5_DPMS.mnu sample file.
BartPE - use .isomemF01 or .isomemwinv file extension
Windows Vista+ Install ISOs (MBR-booting + UEFI-booting) - Convert a working bootable USB drive or ISO to a .imgPTN file. Copy the .imgPTN file to any of the standard menu folders (e.g. \_ISO\MAINMENU or \_ISO\WIN) or to the correct \_ISO\WINDOWS\WINxxx folder)
WinBuilder WinPE ISOs (MBR-booting) - Convert a working bootable USB drive or ISO to a .imgPTN file. Make sure that the correct INI file is on the root of the image.
Hiren's/DLC/DLCD/Strelec ISOs (MBR-booting) - Convert the ISO to a FAT32 .imgPTN file.
WinPE ISOs (MBR-booting) - Use file extension of .ISOPE01 or convert to .imgPTN file if that doesn't work