Overview | Culturing | DNA extraction | DNA quantification | Library preparation | Data analysis | Laboratory Manual | Costs
Purpose
The primary purposes of sequencing in this protocol are to identify mutations within an MTB isolate’s genome which may contribute to its drug resistance profile. Sequencing may be especially useful when the patient is not responding to the drug regimen assigned to them. This is a pragmatic pipeline, developed to aid clinical decision making on treatment, especially for MDR and XDR patients.
Principle
WGS can be done using a number of platforms, but a small-scale approach is to use the ONT MinION device. A MinION is a pocket-sized device which applies nanopore sequencing technology to nucleic acid analyses, with far reaching applications. A nanopore is a nano-scale hole. In its devices, ONT passes an ionic current through nanopores and measures the changes in current as biological molecules (the DNA) pass through the nanopore. The information about the change in current can be used to identify that molecule. The overlapping identical sections of these strings of bases can then be lined up, so that the whole genome can be predicted. This can then be compared to a reference genome and differences mapped. To watch ONT’s explanatory videos, follow this link: https://nanoporetech.com/how-it-works.
Rapid Barcoding kit 96 (SQK-RBK110.96)
More information about this specific kit can be found here: https://store.nanoporetech.com/uk/rapid-barcoding-kit-1.html. The kit is optimised for simplicity and speed, rather than for obtaining maximum throughput. Due to the simple nature of the workflow and the fact that little sample manipulation is required (e.g. minimal pipetting steps and no clean-ups), some very long reads (up to 100 kb; however, the average usually around 10-15 kb) can be achieved with this kit, despite the required transposase fragmentation. However, in order for long reads to be observed in sequencing, long fragments need to be present in the sample in the first place.
This kit and protocol is recommended for users who:
- wish to sequence purified (bacterial) pathogen DNA
- wish to multiplex samples to reduce price per sample
- need a PCR-free method of multiplexing to preserve additional information such as base modifications
- require a short preparation time
- have limited access to laboratory equipment
- have ~400 ng of DNA in each sample
Using high quality native DNA is required for MTB ONT sequencing, as PCR amplifying of MTB does not provide even coverage and results in poor or no depth in some areas, which would result in missing data and potentially incorrect results.
Using ONT to sequence MTB
Sequencing can be done using a number of platforms, but a small-scale approach is to use the ONT MinION device. A nanopore is a nano-scale hole. In its devices, ONT passes an ionic current through nanopores and measures the changes in current as biological molecules (the DNA) pass through the nanopore or near it. The information about the change in current can be used to identify that molecule (e.g. A, T, G or C). The overlapping identical sections of these strings of bases can then be lined up, so that the whole genome can be predicted. This can then be compared to a reference genome and differences mapped. For more information on the requirements of ONT sequencing, please refer to the PANDORA-ID-NET TGHN bioinformatics hub page.