Some types of payload files need to be contiguous in order to work correctly (this applies to all types of E2B USB drives - Removable UFD or Fixed-disk). E2B will warn you if they are not contiguous when you try to boot from them, but they may still boot correctly.
Linux ISOs, linux persistence files (e.g. \ubuntu-rw) and .imgPTN files usually need to be contiguous.
Most Windows Install and WinPE ISOs, .WIM and .VHD files do not need to be contiguous.
E2B can also boot some linux ISOs that it recognises by name, without needing them to be contiguous (via ISOBOOT). If an .ISO file is below 500MB (default limit) and is not contiguous, then E2B will try to copy the contents to a contiguous file (\_ISO\CONTIG.ISO) first so that it can be booted successfully. This can be quite slow! For more details about CONTIG.ISO, see here.
To avoid any 'not contiguous' error messages from E2B, double-click on the \MAKE_THIS_DRIVE_CONTIGUOUS.cmd file on the E2B USB drive to run WinContig and make all E2B files in \ and under \_ISO on the USB drive contiguous - OR - run RMPrepUSB - CTRL+F2 to run WinContig on the whole drive instead.
Tip: You can copy the *.cmd files from \_ISO\docs\WINCONTIG to the root of the E2B USB drive if you wish. Then you can double-click on any one of them to run them.
\MAKE_THIS_DRIVE_CONTIGUOUS.cmd (works on \_ISO and files in \ - i.e. runs WinContig twice)
Files that do not need to be contiguous
Windows Install ISOs, .VHD, .VHDX or .WIM files do not usually need to be contiguous (unless heavily fragmented), and you can just ignore the warning if WinContig cannot make them contiguous.
Also, WinPE .ISO files do not normally need to be contiguous. Test them using E2B and simply press ENTER until they boot. If they boot successfully, you can give the filename a NCQ suffix (e.g. Win10PESE_x64ncq.iso) and this will suppress any E2B 'not contiguous' warnings (E2B v1.98+).
Problems making a large file contiguous
WinContig needs a large amount of contiguous free space and this may not always be available.
You may be able to make an individual file contiguous using Defraggler.
Defraggler can also consolidate free space on a USB drive (Action - Advanced - defrag free space), but that action can also make some contiguous files non-contiguous.