How to make a BioLinux Live USB Stick – with persistent data storage

These are the steps I used to create a batch of bootable BioLinux Live USB sticks – with persistent data so that any data files created/downloaded would be preserved. This was used for a course so that each stick had the same NGS data and the same additional (non-BioLinux) programs pre-installed and already configured.

Step 1 – Download the BioLinux ISO file for use with DVD/USB media

The downloaded .iso file is an archive file that contains the whole BioLinux operating system – it can be used later to either install BioLinux onto a machine, or to create a bootable BioLinux USB Live disk. The bio-linux-8-latest.iso image is currently (March 2016) 3.58GB in size.

Step 2 – Download and install UNetbootin

UNetbootin allows you to create bootable Live USB drives for Ubuntu and other Linux distributions without burning a CD.

It is simple to install, on a Mac you just move the downloaded unetbootin.app file into /Applications

Step 3 – Create an initial BioLinux Live USB disk with persistent data

As the .iso file is 3.58GB in size, a USB stick of atleast 4GB is needed, but that is a little to close for comfort, so best to go for a USB stick of atleast 8GB; these days 8GB sticks are very cheap (£2.99) and are the same price (if not cheaper) as 4GB sticks. To play safe, the USB stick should probably be in FAT32 format – FAT32 has a limitation of 4GB for file sizes – this includes the overall casper-rw BioLinux file which will be where all the persistent data is stored, so if you are going to be storing more than 4GB of data then you will probably need the NTFS file system on the USB stick.

Insert your blank USB key into your computer. Launch unetbootin. Select the “Diskimage” toggle button, select “ISO” from the drop down list, and then navigate to and select the BioLinux .iso file from your computer downloaded in Step 1. Next, in the field entitled “Space used to preserve files across reboots (Ubuntu only)” enter “3500” into the MB textfield (3.5 GB) – you could increase this above 4GB if you have a bigger USB stick and if it is using the NTFS file system. Next, select “USB Drive” from the “Type” drop down list, and then select your actual USB stick from the “Drive” drop down list and then click “OK” to create your bootable BioLinux Live USB stick with persistent data storage.

Step 4 – Boot into your BioLinux

Next step is to boot into the BioLinux Live USB disk from a machine – this will need to be a Windows or Linux machine, a modern Mac is unlikely to boot up from it. Turn the computer off, insert the BioLinux Live USB stick into the computer, turn the computer back on, and get ready. As soon as the first screen appears – which normally has the computer manufacturer logo – it should say something like “Press F12 to Choose Boot Device” at the bottom of the screen – so press F12 quickly before the screen disappears. Sometimes it is not F12, sometimes it is F10 or F2 or another key, but it should say on the screen what button to press. This will launch the BIOS menu. Enter the “Boot Device Select” menu, and move your USB Stick up the boot order to the top, so that the computer will now boot from the USB stick before its own hard drive. Exit the BIOS menu, saving any changes, and the computer should now boot into the BioLinux Live USB stick.

Step 5 – Customise your BioLinux – add data and programs

Now you will be inside your own BioLinux OS on the USB stick. So install any extra programs you want, configure PATHs, and download any data files you want. The programs, configs and data will be saved onto the USB stick and preserved – due to the persistent data storage and the casper-rw file.

Now shutdown BioLinux, remove the USB stick, and boot back into your normal operating system.

Step 6 – Make an image copy of your customised BioLinux disk

Once inside your normal operating system, insert the BioLinux USB stick back in. The next step will only work on a Mac or a Linux machine as it using the dd command.

This copies the BioLinux Live USB stick (located at /dev/disk2 on my machine – on a mac run “diskutil list” to see where yours is) and it creates a single biolinux.img file in the Documents folder which contains the entire operating system along with all the extra data and programs I installed.

The original customised BioLinux Live USB stick can now be ejected and removed.

Step 7 – Copy Copy Copy

Insert a new blank USB stick into the computer (obviously it needs to be atleast the same size as the original one). Now we want to make a copy of that original BioLinux Live USB stick onto the new USB stick using the dd command:

This copies the biolinux.img file located in the Documents folder that we created in Step 6, onto the new blank USB disk (located at /dev/disk2 – check where yours is). On a Mac, I had to first go into DiskUtility and dismount the FAT32 partition of the USB stick before dd would work – not dismount the USB stick itself, just the FAT32 partition. The key thing here, is that you can insert multiple blank USB sticks into all the available USB sticks and run the dd command in parallel:

For an 8GB USB stick, this copying process took almost exactly 1 hour. Then you can eject the USB sticks and put new ones in and copy another batch.

Acknowledgements

Many thanks to Paul Capewell and Willie Weir for a tip on the dd command.