TABLE 5.

Challenges for the development of our rapid TB diagnostic workflow

Challenge	Issue	Solutiona
Sample processing	Insufficient mycobacterial DNA	Differential lysis of human/TB cells to maximize mycobacterial DNA extracted
Library prepn	Time/cost	Modify RPA primers with ONT proprietary chemistry to use ONT PCR sequencing kit
	Time/cost	Freeze-dry RPA primers and reagents to multiplex RPA assay
Sequencing	Cost/hardware	ONT SmidgION, which requires mobile phone rather than laptop/PC
	Cost	ONT Flongle flow cell, which has significantly lower costs than standard MinION flow cells
Scalability	Limited gene panel	Develop RPA primers for additional genes of interest

^aONT, Oxford Nanopore Technologies; RPA, recombinase polymerase amplification; Flongle, trade mark based on “flow cell dongle.”